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Installation

| MARVEL is available on CRAN and also on Github. To access features in beta-testing phase, please install the package from Github: https://github.com/wenweixiong/MARVEL.

Introduction

| This tutorial demonstrates the application of MARVEL for integrated gene and splicing analysis of single-cell RNA-sequencing data. The dataset used to demonstrate the utility of MARVEL here includes induced pluripotent stem cells (iPSCs) and iPSC-induced endoderm cells (Linker et al., 2019). For conciseness, only a subset of the original data will be used here, and only the most salient functions will be demonstrated here. For the complete functionalities of MARVEL, please refer to https://wenweixiong.github.io/MARVEL_Plate.html and https://wenweixiong.github.io/MARVEL_Droplet.html.

Load package

# Load MARVEL package
library(MARVEL)

# Load adjunct packages for this tutorial
library(ggplot2)
library(gridExtra)
# Load adjunct packages to support additional functionalities
library(AnnotationDbi) # GO analysis
library(clusterProfiler)
library(org.Hs.eg.db)
library(org.Mm.eg.db)
# Load adjunct packages to support additional functionalities
library(plyr) # General data processing
library(ggrepel) # General plotting
library(parallel) # To enable multi-thread during RI PSI computation
library(textclean) # AFE, ALE detection
library(fitdistrplus) # Modality analysis: Fit beta distribution
library(FactoMineR) # PCA: Reduce dimension
library(factoextra) # PCA: Retrieve eigenvalues
library(kSamples) # Anderson-Darling (AD) statistical test
library(twosamples) # D Test Statistic (DTS) statistical test
library(stringr) # Plot GO results

Input files

| The input files have been saved in a MARVEL object, and will be elaborated below.

# Load saved MARVEL object
marvel.demo <- readRDS(system.file("extdata/data", "marvel.demo.rds", package="MARVEL"))
class(marvel.demo)
## [1] "Marvel"

Sample metadata

| This is a tab-delimited file created by the user whereby the rows represent the sample (cell) IDs and columns represent the cell information such as cell type, donor ID etc.. Compulsory column is sample.id while all other columns are optional.

SplicePheno <- marvel.demo$SplicePheno
head(SplicePheno)
##     sample.id cell.type sample.type qc.seq
## 2  ERR1562084      iPSC Single Cell   pass
## 10 ERR1562092      iPSC Single Cell   pass
## 18 ERR1562100      iPSC Single Cell   pass
## 25 ERR1562107      iPSC Single Cell   pass
## 38 ERR1562120      iPSC Single Cell   pass
## 41 ERR1562123      iPSC Single Cell   pass

Splice junction counts matrix

| The rows of this matrix represent the splice junction coordinates, the columns represent the sample IDs, and the values represent the splice junction counts. The first column should be named coord.intron. | Here, the splice junction counts were quantified using the STAR aligner version 2.6.1d in 2-pass mode (Dobin et al., 2013). An example code for one sample (ERR1562083) below. Note a separate folder SJ is created here to contain the splice junction count files (SJ.out.tab) generated from 1st pass mode to be used for 2nd pass mode.

# STAR in 1st pass mode
STAR --runThreadN 16 \
     --genomeDir GRCh38_GENCODE_genome_STAR_indexed \
     --readFilesCommand zcat \
     --readFilesIn ERR1562083_1_val_1.fq.gz ERR1562083_2_val_2.fq.gz \
     --outFileNamePrefix SJ/ERR1562083. \
     --outSAMtype None

# STAR in 2nd pass mode
STAR --runThreadN 16 \
     --genomeDir GRCh38_GENCODE_genome_STAR_indexed \
     --readFilesCommand zcat \
     --readFilesIn ERR1562083_1_val_1.fq.gz ERR1562083_2_val_2.fq.gz \
     --outFileNamePrefix ERR1562083. \
     --sjdbFileChrStartEnd SJ/*SJ.out.tab \
     --outSAMtype BAM SortedByCoordinate \
     --outSAMattributes NH HI AS nM XS \
     --quantMode TranscriptomeSAM

| Once the individual splice junction count files have been generated, they should be collated and read into R as follows:

SpliceJunction <- marvel.demo$SpliceJunction
SpliceJunction[1:5,1:5]
##                 coord.intron ERR1562084 ERR1562092 ERR1562100 ERR1562107
## 44383 chr1:19357564:19378539        219         37        457        276
## 44387 chr1:19374456:19378539         NA         NA         NA         NA
## 71516 chr1:28236453:28237760          5         22         58         16
## 71517 chr1:28236453:28237836        193        104        265        190
## 93429 chr1:62196529:62203446          4         NA         13         NA

Splicing event metadata

| The rows of this metadata represent the splicing events while the columns represent the splicing event information such as the transcript ID and the corresponding gene information. Compulsory columns are tran_id and gene_id. | The splicing events here were detected using rMATS version 4.1.0 (Shen et al., 2014). For preparing splicing event nomenclatures (tran_id), please refer to https://wenweixiong.github.io/Splicing_Nomenclature. Example code for running rMATS as follows. | Note that any BAM files may be specified in --b1 and --b2. This is because rMATS requires these specification for statistical testing of splicing events between the two samples. But here, we will only be using the splicing events detected (fromGTF.SE.txt, fromGTF.MXE.txt, fromGTF.RI.txt, fromGTF.A5SS.txt, fromGTF.A3SS.txt), but not the statistical test results, from this step for our downstream analysis.

rmats \
    --b1 path_to_BAM_sample_1.txt \
    --b2 path_to_BAM_sample_2.txt \
    --gtf gencode.v31.annotation.gtf \
    --od rMATS/ \
    --tmp rMATS/ \
    -t paired \
    --readLength 125 \
    --variable-read-length \
    --nthread 8 \
    --statoff

| Once the individual splicing event files for SE, MXE, RI, A5S5, and A3SS have been generated, they may be read into R as follows:

SpliceFeature <-marvel.demo$SpliceFeature
lapply(SpliceFeature, head)
## $SE
##                                                                           tran_id
## 9578     chr1:62196435:62196528:+@chr1:62203447:62203557:+@chr1:62206519:62207636
## 10483 chr15:24962114:24962209:+@chr15:24967029:24967152:+@chr15:24967932:24968082
## 10484 chr15:24962114:24962209:+@chr15:24967029:24967240:+@chr15:24967932:24968082
## 11921 chr17:51153576:51153662:+@chr17:51154225:51154444:+@chr17:51155651:51155780
## 15780 chr10:78037194:78037304:+@chr10:78037439:78037441:+@chr10:78040204:78040225
## 15782 chr10:78037194:78037304:+@chr10:78037965:78037982:+@chr10:78040204:78040225
##                  gene_id gene_short_name      gene_type
## 9578   ENSG00000240563.2           L1TD1 protein_coding
## 10483 ENSG00000128739.22           SNRPN protein_coding
## 10484 ENSG00000128739.22           SNRPN protein_coding
## 11921  ENSG00000239672.7            NME1 protein_coding
## 15780 ENSG00000138326.20           RPS24 protein_coding
## 15782 ENSG00000138326.20           RPS24 protein_coding
## 
## $MXE
##                                                                                                        tran_id
## 541      chr14:22830187:22830254:+@chr14:22830964:22831037:+@chr14:22833418:22833482:+@chr14:22834172:22835037
## 1402     chr11:62761795:62761904:+@chr11:62762542:62762866:+@chr11:62762867:62763026:+@chr11:62765182:62765232
## 1589 chr7:128748573:128748804:+@chr7:128754262:128754455:+@chr7:128754529:128754722:+@chr7:128758871:128759037
## 1792                         chr2:271866:271939:+@chr2:272037:272150:+@chr2:272192:272305:+@chr2:275140:275201
## 2221 chr3:186786164:186786273:+@chr3:186786418:186786465:+@chr3:186786502:186786645:+@chr3:186787127:186787264
## 2262     chr12:98593979:98594135:+@chr12:98595433:98595661:+@chr12:98595727:98595848:+@chr12:98597856:98598035
##                 gene_id gene_short_name      gene_type
## 541  ENSG00000172590.18          MRPL52 protein_coding
## 1402 ENSG00000168002.12          POLR2G protein_coding
## 1589 ENSG00000128595.17            CALU protein_coding
## 1792 ENSG00000143727.16            ACP1 protein_coding
## 2221 ENSG00000156976.17          EIF4A2 protein_coding
## 2262 ENSG00000075415.12         SLC25A3 protein_coding
## 
## $RI
##                                                tran_id            gene_id
## 776    chr6:34236873:34236963:+@chr6:34237204:34237317 ENSG00000137309.19
## 4207 chr13:43055391:43058296:+@chr13:43058654:43058862  ENSG00000120675.6
## 4209 chr13:43059394:43059714:+@chr13:43062190:43062295  ENSG00000120675.6
## 5511   chr2:55232808:55232872:+@chr2:55233363:55233417 ENSG00000143947.13
## 6845       chr7:5528719:5528564:-@chr7:5528344:5528281 ENSG00000075624.16
## 6927   chr6:73519228:73519128:-@chr6:73519064:73518932 ENSG00000156508.18
##      gene_short_name      gene_type
## 776            HMGA1 protein_coding
## 4207         DNAJC15 protein_coding
## 4209         DNAJC15 protein_coding
## 5511          RPS27A protein_coding
## 6845            ACTB protein_coding
## 6927          EEF1A1 protein_coding
## 
## $A5SS
##                                                              tran_id
## 493    chr7:141738358:141738410|141738503:+@chr7:141739124:141739190
## 2635      chr19:41860255:41860289|41860445:+@chr19:41860775:41860845
## 4148   chr3:197950190:197950221|197950299:+@chr3:197950936:197950978
## 4721   chr8:144792587:144792245|144792366:-@chr8:144791992:144792140
## 5174        chr6:34426071:34425796|34426032:-@chr6:34425072:34425221
## 5878 chr12:112409641:112409411|112409587:-@chr12:112408420:112408656
##                 gene_id gene_short_name      gene_type
## 493  ENSG00000106028.11           SSBP1 protein_coding
## 2635  ENSG00000105372.7           RPS19 protein_coding
## 4148 ENSG00000182899.17          RPL35A protein_coding
## 4721 ENSG00000161016.17            RPL8 protein_coding
## 5174  ENSG00000270800.3     RPS10-NUDT3 protein_coding
## 5878 ENSG00000089009.15      AC004086.1 protein_coding
## 
## $A3SS
##                                                            tran_id
## 1411      chr1:28236361:28236452:+@chr1:28237761|28237837:28238105
## 1541              chr16:399627:399739:+@chr16:400109|400219:400754
## 2351      chr1:62196435:62196528:+@chr1:62203447|62206519:62207636
## 3961    chr10:78037194:78037304:+@chr10:78037439|78040204:78040225
## 4321    chr12:56158684:56158711:+@chr12:56159587|56159975:56160148
## 7009 chr9:136862906:136862999:-@chr9:136862345|136862496:136862119
##                 gene_id gene_short_name      gene_type
## 1411 ENSG00000130770.18         ATP5IF1 protein_coding
## 1541 ENSG00000103202.13            NME4 protein_coding
## 2351  ENSG00000240563.2           L1TD1 protein_coding
## 3961 ENSG00000138326.20           RPS24 protein_coding
## 4321 ENSG00000092841.18            MYL6 protein_coding
## 7009 ENSG00000107223.13            EDF1 protein_coding
## 
## $ALE
##                                                                        tran_id
## 227    chr3:184366717:184366800:+@chr3:184368113:184368596|184368177:184368287
## 311          chr6:41789232:41789336:+@chr6:41789531:41790141|41789568:41789891
## 449        chr10:78037194:78037304:+@chr10:78037965:78038104|78040615:78040696
## 461  chr10:123162612:123162828:+@chr10:123163820:123170467|123165047:123165365
## 694                    chr16:399627:399739:+@chr16:400109:400648|400219:400416
## 1037         chr1:19378540:19378653:-@chr1:19374175:19374455|19357522:19357563
##      event_type            gene_id gene_short_name      gene_type
## 227         ALE  ENSG00000163882.9          POLR2H protein_coding
## 311         ALE ENSG00000124593.16      AL365205.1 protein_coding
## 449         ALE ENSG00000138326.20           RPS24 protein_coding
## 461         ALE ENSG00000154473.18            BUB3 protein_coding
## 694         ALE ENSG00000103202.13            NME4 protein_coding
## 1037        ALE ENSG00000077549.18           CAPZB protein_coding
## 
## $AFE
##                                                                      tran_id
## 739  chr4:108620569:108620600|108620656:108620712:+@chr4:108621951:108622024
## 1806     chr13:27251561:27251585|27251597:27251699:+@chr13:27253765:27253843
## 3775 chr5:150449703:150449739|150449492:150449696:-@chr5:150447585:150447735
## 3776 chr5:150449703:150449739|150449486:150449691:-@chr5:150447585:150447735
## 3862       chr6:34426032:34426052|34425796:34426026:-@chr6:34425072:34425221
## 4208 chr8:144792366:144792423|144792245:144792389:-@chr8:144791992:144792140
##      event_type            gene_id gene_short_name      gene_type
## 739         AFE ENSG00000109475.16           RPL34 protein_coding
## 1806        AFE ENSG00000122026.10           RPL21 protein_coding
## 3775        AFE ENSG00000164587.13           RPS14 protein_coding
## 3776        AFE ENSG00000164587.13           RPS14 protein_coding
## 3862        AFE  ENSG00000270800.3     RPS10-NUDT3 protein_coding
## 4208        AFE ENSG00000161016.17            RPL8 protein_coding

Intron count matrix

| The rows of this matrix represent intron coordinates, the columns represent the sample IDs, and the values represent the total reads mapping to the intron. These values will be used to compute the percent spliced-in (PSI) values of retained introns (RI) splicing events downstream. | Here, intron coverage was computed using Bedtools version 2.27.1 (Quinlan et al., 2010). Example code for one sample (ERR1562083) below. This code computes the counts at each base of a given intron, the sum of which, will be the total counts for the given intron. It is this total counts that is represented in the matrix. | Note for GRCh38.primary_assembly.genome_bedtools.txt, the first column consists of the chromosome name (chr1, chr2, chr3…) and the second column consists of the chromosome size or length. Additionally, the BED file RI_Coordinates.bed contains the intron coordinates from RI_featureData.txt generated from rMATS in the previous step.

bedtools coverage \
               -g GRCh38.primary_assembly.genome_bedtools.txt \
               -split \
               -sorted \
               -a RI_Coordinates.bed \
               -b ERR1562083.Aligned.sortedByCoord.out.bam > \
                  ERR1562083.txt \
               -d

| Once the individual splice junction count files have been generated, they should be collated and read into R as follows:

IntronCounts <- marvel.demo$IntronCounts
IntronCounts[1:5,1:5]
##                coord.intron ERR1562084 ERR1562092 ERR1562100 ERR1562107
## 1233 chr2:55232873:55233362        259        141        481        175
## 2562 chr4:39458442:39458890       5466       6105      46238      20963
## 3552 chr6:34236964:34237203       2395         30       2940        547
## 3657 chr6:73519065:73519127     117305      69093     204019     123522
## 3867   chr7:5528345:5528563     231508     146758     458847     297025

Gene expression matrix

| The rows of this matrix represent the gene IDs, the columns represent the sample IDs, and the values represent the normalised gene expression counts (e.g., RPKM/FPKM/TPM), but not yet log2-transformed. | Here, gene expression was quantified using RSEM version 1.2.31 (Li et al., 2011). Example code for one sample (ERR1562083) as follows. Here, the values returned are in transcript per million (TPM) unit.

rsem-calculate-expression --bam \
                          --paired-end \
                          -p 8 \
                          ERR1562083.Aligned.toTranscriptome.out.bam \
                          GRCh38_GENCODE_genome_RSEM_indexed/gencode.v31 \
                          ERR1562083

| Once the individual gene expression files have been generated, they should be collated and read into R as follows:

Exp <- marvel.demo$Exp
Exp[1:5,1:5]
##                 gene_id ERR1562084 ERR1562092 ERR1562100 ERR1562107
## 1007 ENSG00000067225.18   8.007924   5.993674   7.876640   7.956231
## 1248 ENSG00000073921.18   3.460743   6.365798   4.032101   7.397974
## 1309 ENSG00000075415.12   9.497652   9.838385   8.908573   9.913742
## 1317 ENSG00000075624.16  11.493971  11.368496  11.589609  11.712037
## 1392 ENSG00000077549.18   8.425887   6.874797   8.566244   8.752314

Gene metadata

| The rows of this metadata represent the gene IDs while the columns represent the gene information such as the abbreviated gene names and gene type. Compulsory columns are gene_id, gene_short_name, and gene_type. All other columns are optional. | Here, the metadata information was parsed and retrieved from gencode.v31.annotation.gtf.

GeneFeature <- marvel.demo$GeneFeature
head(GeneFeature)
##                 gene_id gene_short_name      gene_type
## 1007 ENSG00000067225.18             PKM protein_coding
## 1248 ENSG00000073921.18          PICALM protein_coding
## 1309 ENSG00000075415.12         SLC25A3 protein_coding
## 1317 ENSG00000075624.16            ACTB protein_coding
## 1392 ENSG00000077549.18           CAPZB protein_coding
## 1806 ENSG00000089009.15      AC004086.1 protein_coding

Create MARVEL object

marvel <- CreateMarvelObject(SpliceJunction=SpliceJunction,
                             SplicePheno=SplicePheno,
                             SpliceFeature=SpliceFeature,
                             IntronCounts=IntronCounts,
                             GeneFeature=GeneFeature,
                             Exp=Exp
                             )

Compute PSI

| MARVEL will compute the percent spliced-in (PSI) values for each splicing event. Only splicing event supported by splice junction reads, i.e., high-confidence splicing events, will be selected for PSI quantification. The minimum number of splice junction reads required may be specified using the CoverageThreshold option.

plot of chunk unnamed-chunk-17

| PSI is simply the proportion of reads supporting the inclusion of the alternative exon divided by the total number of reads mapping to the splicing event, which encompasses the reads supporting the inclusion and also reads supporting the exclusion of the splicing event. This fraction is in turn converted to percentage.

plot of chunk unnamed-chunk-18

# Check splicing junction data
marvel.demo <- CheckAlignment(MarvelObject=marvel.demo, level="SJ")
# Validate, filter, compute SE splicing events
marvel.demo <- ComputePSI(MarvelObject=marvel.demo,
                          CoverageThreshold=10,
                          UnevenCoverageMultiplier=10,
                          EventType="SE"
                          )

# Validate, filter, compute MXE splicing events    
marvel.demo <- ComputePSI(MarvelObject=marvel.demo,
                          CoverageThreshold=10,
                          UnevenCoverageMultiplier=10,
                          EventType="MXE"
                          )

# Validate, filter, compute RI splicing events      
marvel.demo <- ComputePSI(MarvelObject=marvel.demo,
                          CoverageThreshold=10,
                          EventType="RI",
                          thread=4
                          )

# Validate, filter, compute A5SS splicing events  
marvel.demo <- ComputePSI(MarvelObject=marvel.demo,
                          CoverageThreshold=10,
                          EventType="A5SS"
                          )

# Validate, filter, compute A3SS splicing events  
marvel.demo <- ComputePSI(MarvelObject=marvel.demo,
                          CoverageThreshold=10,
                          EventType="A3SS"
                          )

# Validate, filter, compute AFE splicing events     
marvel.demo <- ComputePSI(MarvelObject=marvel.demo,
                          CoverageThreshold=10,
                          EventType="AFE"
                          )

# Validate, filter, compute ALE splicing events      
marvel.demo <- ComputePSI(MarvelObject=marvel.demo,
                          CoverageThreshold=10,
                          EventType="ALE"
                          )

| The common option across all functions for computing PSI value is CoverageThreshold. This option indicates the minimum number of splice junction reads supporting the splicing events, above which, the PSI will be computed. PSI of splicing events below this threshold will be coded as NA. | Options specific to a given splicing event are:

Pre-flight check

| This step ensures that our data is ready for further downstream analysis, including modality assignment, differential expression analysis, dimension reduction, and functional annotation.

Transform expression values

| Gene expression values will be log2-transformed. You may skip this step if your gene expression matrix has been transformed prior to creating the MARVEL object.

marvel.demo <- TransformExpValues(MarvelObject=marvel.demo,
                                  offset=1,
                                  transformation="log2",
                                  threshold.lower=1
                                  )

Check matrices and metadata

| We will have to make sure the columns of the matrices align with the sample IDs of the sample metadata and the rows of the matrices align with the feature metadata. Finally, the columns across all matrices should align with one another.

# Check splicing data
marvel.demo <- CheckAlignment(MarvelObject=marvel.demo, level="splicing")

# Check gene data
marvel.demo <- CheckAlignment(MarvelObject=marvel.demo, level="gene")

# Cross-check splicing and gene data
marvel.demo <- CheckAlignment(MarvelObject=marvel.demo, level="splicing and gene")

| Our data is ready for downstream analysis when only MATCHED flags are reported. If any NOT MATCHED flags are reported, please double-check the input file requirements.

Overview of splicing events

| Let's have an overview of the number of splicing events expressed in a given cell population, and stratify them by splicing event type.

iPSC

# Retrieve sample metadata
df.pheno <- marvel.demo$SplicePheno

# Define sample ids
sample.ids <- df.pheno[which(df.pheno$cell.type=="iPSC"), "sample.id"]

# Tabulate expressed events
marvel.demo <- CountEvents(MarvelObject=marvel.demo,
                           sample.ids=sample.ids,
                           min.cells=5
                           )

# Output (1): Plot
marvel.demo$N.Events$Plot

plot of chunk unnamed-chunk-23

# Output (2): Table
marvel.demo$N.Events$Table
##   event_type freq      pct
## 1         SE   10 14.28571
## 2        MXE   10 14.28571
## 3         RI   10 14.28571
## 4       A5SS   10 14.28571
## 5       A3SS   10 14.28571
## 6        ALE   10 14.28571
## 7        AFE   10 14.28571

Endoderm cells

# Retrieve sample metadata
df.pheno <- marvel.demo$SplicePheno

# Define sample ids
sample.ids <- df.pheno[which(df.pheno$cell.type=="Endoderm"), "sample.id"]

# Tabulate expressed events
marvel.demo <- CountEvents(MarvelObject=marvel.demo,
                           sample.ids=sample.ids,
                           min.cells=5
                           )

# Output (1): Plot
marvel.demo$N.Events$Plot

plot of chunk unnamed-chunk-24

# Output (2): Table
marvel.demo$N.Events$Table
##   event_type freq      pct
## 1         SE   10 14.28571
## 2        MXE   10 14.28571
## 3         RI   10 14.28571
## 4       A5SS   10 14.28571
## 5       A3SS   10 14.28571
## 6        ALE   10 14.28571
## 7        AFE   10 14.28571

Modality analysis

| The PSI distribution for a given splicing event in a given cell population may be assigned to a modality class. Modalities are simply discrete splicing patterns categories. This will enable us to understand the isoform expression pattern for a given splicing event in a given cell population. | The five main modalities are included, excluded, bimodal, middle, and multimodal (Song et al., 2017). MARVEL provides finer classification of splicing patterns by further stratifying included and excluded modalities into primary and dispersed.

plot of chunk unnamed-chunk-25

iPSC

# Retrieve sample metadata
df.pheno <- marvel.demo$SplicePheno

# Define sample IDs
sample.ids <- df.pheno[which(df.pheno$cell.type=="iPSC"), "sample.id"]

# Assign modality
marvel.demo <- AssignModality(MarvelObject=marvel.demo,
                              sample.ids=sample.ids,
                              min.cells=5,
                              seed=1
                              )

marvel.demo$Modality$Results[1:5, c("tran_id", "event_type", "gene_id", "gene_short_name", "modality.bimodal.adj")]
##                                                                       tran_id
## 1    chr1:62196435:62196528:+@chr1:62203447:62203557:+@chr1:62206519:62207636
## 2 chr15:24962114:24962209:+@chr15:24967029:24967152:+@chr15:24967932:24968082
## 3 chr15:24962114:24962209:+@chr15:24967029:24967240:+@chr15:24967932:24968082
## 4 chr17:51153576:51153662:+@chr17:51154225:51154444:+@chr17:51155651:51155780
## 5 chr10:78037194:78037304:+@chr10:78037439:78037441:+@chr10:78040204:78040225
##   event_type            gene_id gene_short_name modality.bimodal.adj
## 1         SE  ENSG00000240563.2           L1TD1     Excluded.Primary
## 2         SE ENSG00000128739.22           SNRPN   Excluded.Dispersed
## 3         SE ENSG00000128739.22           SNRPN   Excluded.Dispersed
## 4         SE  ENSG00000239672.7            NME1   Excluded.Dispersed
## 5         SE ENSG00000138326.20           RPS24   Excluded.Dispersed
# Tabulate modality proportion (overall)
marvel.demo <- PropModality(MarvelObject=marvel.demo,
                            modality.column="modality.bimodal.adj",
                            modality.type="extended",
                            event.type=c("SE", "MXE", "RI", "A5SS", "A3SS", "AFE", "ALE"),
                            across.event.type=FALSE
                            )

marvel.demo$Modality$Prop$DoughnutChart$Plot

plot of chunk unnamed-chunk-26

marvel.demo$Modality$Prop$DoughnutChart$Table
##             modality freq       pct
## 4   Included.Primary    2  2.857143
## 3 Included.Dispersed    6  8.571429
## 2   Excluded.Primary   18 25.714286
## 1 Excluded.Dispersed   39 55.714286
## 5             Middle    2  2.857143
## 6         Multimodal    3  4.285714
# Tabulate modality proportion (by event type)
marvel.demo <- PropModality(MarvelObject=marvel.demo,
                            modality.column="modality.bimodal.adj",
                            modality.type="extended",
                            event.type=c("SE", "MXE", "RI", "A5SS", "A3SS", "AFE", "ALE"),
                            across.event.type=TRUE,
                            prop.test="fisher",
                            prop.adj="fdr",
                            xlabels.size=8
                            )

marvel.demo$Modality$Prop$BarChart$Plot

plot of chunk unnamed-chunk-27

head(marvel.demo$Modality$Prop$BarChart$Table)
##                modality freq pct event_type
## 3  Included (Dispersed)    1  10         SE
## 2    Excluded (Primary)    2  20         SE
## 1  Excluded (Dispersed)    6  60         SE
## 4                Middle    1  10         SE
## 11           Multimodal    0   0         SE
## 21   Included (Primary)    0   0         SE

Endoderm

# Retrieve sample metadata
df.pheno <- marvel.demo$SplicePheno

# Define sample IDs
sample.ids <- df.pheno[which(df.pheno$cell.type=="Endoderm"), "sample.id"]

# Assign modality
marvel.demo <- AssignModality(MarvelObject=marvel.demo,
                              sample.ids=sample.ids,
                              min.cells=5,
                              seed=1
                              )

marvel.demo$Modality$Results[1:5, c("tran_id", "event_type", "gene_id", "gene_short_name", "modality.bimodal.adj")]
##                                                                       tran_id
## 1    chr1:62196435:62196528:+@chr1:62203447:62203557:+@chr1:62206519:62207636
## 2 chr15:24962114:24962209:+@chr15:24967029:24967152:+@chr15:24967932:24968082
## 3 chr15:24962114:24962209:+@chr15:24967029:24967240:+@chr15:24967932:24968082
## 4 chr17:51153576:51153662:+@chr17:51154225:51154444:+@chr17:51155651:51155780
## 5 chr10:78037194:78037304:+@chr10:78037439:78037441:+@chr10:78040204:78040225
##   event_type            gene_id gene_short_name modality.bimodal.adj
## 1         SE  ENSG00000240563.2           L1TD1   Excluded.Dispersed
## 2         SE ENSG00000128739.22           SNRPN     Excluded.Primary
## 3         SE ENSG00000128739.22           SNRPN   Excluded.Dispersed
## 4         SE  ENSG00000239672.7            NME1     Excluded.Primary
## 5         SE ENSG00000138326.20           RPS24   Excluded.Dispersed
# Tabulate modality proportion (overall)
marvel.demo <- PropModality(MarvelObject=marvel.demo,
                            modality.column="modality.bimodal.adj",
                            modality.type="extended",
                            event.type=c("SE", "MXE", "RI", "A5SS", "A3SS", "AFE", "ALE"),
                            across.event.type=FALSE
                            )

marvel.demo$Modality$Prop$DoughnutChart$Plot

plot of chunk unnamed-chunk-28

marvel.demo$Modality$Prop$DoughnutChart$Table
##             modality freq       pct
## 5   Included.Primary    2  2.857143
## 4 Included.Dispersed    7 10.000000
## 3   Excluded.Primary   28 40.000000
## 2 Excluded.Dispersed   29 41.428571
## 1            Bimodal    1  1.428571
## 6             Middle    1  1.428571
## 7         Multimodal    2  2.857143
# Tabulate modality proportion (by event type)
marvel.demo <- PropModality(MarvelObject=marvel.demo,
                            modality.column="modality.bimodal.adj",
                            modality.type="extended",
                            event.type=c("SE", "MXE", "RI", "A5SS", "A3SS", "AFE", "ALE"),
                            across.event.type=TRUE,
                            prop.test="fisher",
                            prop.adj="fdr",
                            xlabels.size=8
                           )

marvel.demo$Modality$Prop$BarChart$Plot

plot of chunk unnamed-chunk-29

head(marvel.demo$Modality$Prop$BarChart$Table)
##                modality freq pct event_type
## 2    Excluded (Primary)    3  30         SE
## 1  Excluded (Dispersed)    5  50         SE
## 3                Middle    1  10         SE
## 4            Multimodal    1  10         SE
## 11 Included (Dispersed)    0   0         SE
## 21              Bimodal    0   0         SE

Differential analysis

| Differential analysis is the cornerstone of RNA-sequencing analysis. This is the first step to identify candidate genes and isoforms for downstream experimental validation. | Statistical tests that compare the mean expression values between two cell populations, such as Wilcox, are suitable for differential gene expression analysis. | However, the mean alone will not be sufficient to detect changes in splicing patterns. For example, based on the mean alone, it may not be possible to distinguish between splicing events with bimodal, middle, and multimodal splicing patterns. Therefore, in lieu of comparing mean, MARVEL compares the overall PSI distribution between two cell populations.

plot of chunk unnamed-chunk-30

Differential gene expression analysis

# Define cell groups
    # Retrieve sample metadata
    df.pheno <- marvel.demo$SplicePheno

    # Cell group 1 (reference)
    cell.group.g1 <- df.pheno[which(df.pheno$cell.type=="iPSC"), "sample.id"]

    # Cell group 2
    cell.group.g2 <- df.pheno[which(df.pheno$cell.type=="Endoderm"), "sample.id"]

# DE analysis
marvel.demo <- CompareValues(MarvelObject=marvel.demo,
                             cell.group.g1=cell.group.g1,
                             cell.group.g2=cell.group.g2,
                             min.cells=3,
                             method="t.test",
                             method.adjust="fdr",
                             level="gene",
                             show.progress=FALSE
                             )

marvel.demo$DE$Exp$Table[1:5, ]
##              gene_id gene_short_name      gene_type n.cells.g1 n.cells.g2
## 1 ENSG00000128739.22           SNRPN protein_coding         15         15
## 2 ENSG00000067225.18             PKM protein_coding         15         15
## 3 ENSG00000137309.19           HMGA1 protein_coding         15         15
## 4  ENSG00000239672.7            NME1 protein_coding         15         13
## 5  ENSG00000120675.6         DNAJC15 protein_coding         14         15
##    mean.g1   mean.g2     log2fc statistic        p.val    p.val.adj
## 1 9.203101  8.331827 -0.8712731  6.773031 9.876425e-07 5.333270e-05
## 2 7.896393 10.207866  2.3114738 -6.477287 2.845515e-06 7.682892e-05
## 3 7.276139  6.060553 -1.2155857  6.148961 5.694655e-06 1.025038e-04
## 4 8.985144  5.082584 -3.9025599  5.627087 5.476249e-05 6.832612e-04
## 5 3.871605  6.065549  2.1939435 -4.706610 6.484850e-05 6.832612e-04

Volcano plot: Genes

# Plot DE results
marvel.demo <- PlotDEValues(MarvelObject=marvel.demo,
                            pval=0.10,
                            log2fc=0.5,
                            point.size=0.1,
                            level="gene.global",
                            anno=FALSE
                            )

marvel.demo$DE$Exp.Global$Plot

plot of chunk unnamed-chunk-32

marvel.demo$DE$Exp.Global$Summary
##    sig freq
## 1   up    4
## 2 down   20
## 3 n.s.   30
head(marvel.demo$DE$Exp.Global$Table[,c("gene_id", "gene_short_name", "sig")])
##              gene_id gene_short_name  sig
## 1 ENSG00000128739.22           SNRPN down
## 2 ENSG00000067225.18             PKM   up
## 3 ENSG00000137309.19           HMGA1 down
## 4  ENSG00000239672.7            NME1 down
## 5  ENSG00000120675.6         DNAJC15   up
## 6 ENSG00000154473.18            BUB3 down
# Plot DE results with annotation of selected genes
    # Retrieve DE output table
    results <- marvel.demo$DE$Exp$Table

    # Retrieve top genes
    index <- which(results$log2fc > 2 | results$log2fc < -2)
    gene_short_names <- results[index, "gene_short_name"]

    # Plot
    marvel.demo <- PlotDEValues(MarvelObject=marvel.demo,
                                pval=0.10,
                                log2fc=0.5,
                                point.size=0.1,
                                xlabel.size=10,
                                level="gene.global",
                                anno=TRUE,
                                anno.gene_short_name=gene_short_names
                                )

    marvel.demo$DE$Exp.Global$Plot

plot of chunk unnamed-chunk-33

Differential splicing analysis

marvel.demo <- CompareValues(MarvelObject=marvel.demo,
                             cell.group.g1=cell.group.g1,
                             cell.group.g2=cell.group.g2,
                             min.cells=5,
                             method=c("ad", "dts"),
                             method.adjust="fdr",
                             level="splicing",
                             event.type=c("SE", "MXE", "RI", "A5SS", "A3SS", "ALE", "AFE"),
                             show.progress=FALSE
                             )

head(marvel.demo$DE$PSI$Table[["ad"]])
##                                                                         tran_id
## 110                       chr17:8383254:8382781|8383157:-@chr17:8382143:8382315
## 210               chr17:8383157:8383193|8382781:8383164:-@chr17:8382143:8382315
## 310 chr15:24962114:24962209:+@chr15:24967029:24967152:+@chr15:24967932:24968082
## 410               chr8:144792587:144792245|144792366:-@chr8:144791992:144792140
## 55      chr8:144792366:144792423|144792245:144792389:-@chr8:144791992:144792140
## 61      chr5:150449703:150449739|150449492:150449696:-@chr5:150447585:150447735
##     event_type            gene_id gene_short_name      gene_type n.cells.g1
## 110       A5SS ENSG00000161970.15           RPL26 protein_coding         15
## 210        AFE ENSG00000161970.15           RPL26 protein_coding         15
## 310         SE ENSG00000128739.22           SNRPN protein_coding         15
## 410       A5SS ENSG00000161016.17            RPL8 protein_coding         15
## 55         AFE ENSG00000161016.17            RPL8 protein_coding         15
## 61         AFE ENSG00000164587.13           RPS14 protein_coding         15
##     n.cells.g2  mean.g1    mean.g2 mean.diff statistic      p.val    p.val.adj
## 110         15 6.663009 0.02754821 -6.635461    18.553 5.1855e-11 1.814925e-09
## 210         15 6.663009 0.02754821 -6.635461    18.553 5.1855e-11 1.814925e-09
## 310         13 8.944493 0.00000000 -8.944493    18.128 8.3362e-11 1.945113e-09
## 410         15 9.446244 0.53253936 -8.913705    16.638 6.7920e-10 9.508800e-09
## 55          15 9.446244 0.53253936 -8.913705    16.638 6.7920e-10 9.508800e-09
## 61          15 3.830668 0.04283629 -3.787831    15.942 1.7288e-09 2.016933e-08
##     modality.bimodal.adj.g1 modality.bimodal.adj.g2 n.cells.outliers.g1
## 110      Excluded.Dispersed        Excluded.Primary                  15
## 210      Excluded.Dispersed        Excluded.Primary                  15
## 310      Excluded.Dispersed        Excluded.Primary                  15
## 410      Excluded.Dispersed        Excluded.Primary                  15
## 55       Excluded.Dispersed        Excluded.Primary                  15
## 61         Excluded.Primary        Excluded.Primary                  15
##     n.cells.outliers.g2 outliers
## 110                   1    FALSE
## 210                   1    FALSE
## 310                   0    FALSE
## 410                   2    FALSE
## 55                    2    FALSE
## 61                    3    FALSE
head(marvel.demo$DE$PSI$Table[["dts"]])
##                                                                         tran_id
## 4   chr10:78037194:78037304:+@chr10:78040204:78040225:+@chr10:78040615:78040747
## 5   chr11:85981129:85981228:-@chr11:85978070:85978093:-@chr11:85976623:85976682
## 18      chr4:108620569:108620600|108620656:108620712:+@chr4:108621951:108622024
## 1                               chr6:73519228:73519128:-@chr6:73519064:73518932
## 110 chr15:24962114:24962209:+@chr15:24967029:24967152:+@chr15:24967932:24968082
## 210 chr17:51153576:51153662:+@chr17:51154225:51154444:+@chr17:51155651:51155780
##     event_type            gene_id gene_short_name      gene_type n.cells.g1
## 4           SE ENSG00000138326.20           RPS24 protein_coding         15
## 5           SE ENSG00000073921.18          PICALM protein_coding          8
## 18         AFE ENSG00000109475.16           RPL34 protein_coding         15
## 1           RI ENSG00000156508.18          EEF1A1 protein_coding         15
## 110         SE ENSG00000128739.22           SNRPN protein_coding         15
## 210         SE  ENSG00000239672.7            NME1 protein_coding         12
##     n.cells.g2   mean.g1   mean.g2  mean.diff  statistic p.val p.val.adj
## 4           15 43.540716 63.151243  19.610527 0.09303941 5e-04     5e-04
## 5            8 90.322395  8.970588 -81.351806 0.41267135 5e-04     5e-04
## 18          15 33.464032  7.113192 -26.350840 0.10657646 5e-04     5e-04
## 1           15 86.866886 82.948010  -3.918876 0.01814701 5e-04     5e-04
## 110         13  8.944493  0.000000  -8.944493 0.03816722 5e-04     5e-04
## 210          9 10.109246  0.000000 -10.109246 0.05011268 5e-04     5e-04
##     modality.bimodal.adj.g1 modality.bimodal.adj.g2 n.cells.outliers.g1
## 4                    Middle                  Middle                   0
## 5        Included.Dispersed      Excluded.Dispersed                   0
## 18                   Middle      Excluded.Dispersed                   0
## 1          Included.Primary        Included.Primary                  15
## 110      Excluded.Dispersed        Excluded.Primary                  15
## 210      Excluded.Dispersed        Excluded.Primary                  11
##     n.cells.outliers.g2 outliers
## 4                     0    FALSE
## 5                     0    FALSE
## 18                    0    FALSE
## 1                    15    FALSE
## 110                   0    FALSE
## 210                   0    FALSE

Distance plot: Splicing

marvel.demo <- PlotDEValues(MarvelObject=marvel.demo,
                       method="ad",
                       pval=0.10,
                       level="splicing.distance",
                       anno=TRUE,
                       anno.tran_id=marvel.demo$DE$PSI$Table[["ad"]]$tran_id[c(1:10)]
                       )

marvel.demo$DE$PSI$Plot[["ad"]]

plot of chunk unnamed-chunk-35

Differential (spliced) gene analysis

| Next, we will perform differential gene expression analysis only on the differentially spliced genes. This will enable us to investigate the gene-splicing relationship between iPSCs and endoderm cells downstream.

marvel.demo <- CompareValues(MarvelObject=marvel.demo,
                             cell.group.g1=cell.group.g1,
                             cell.group.g2=cell.group.g2,
                             psi.method=c("ad", "dts"),
                             psi.pval=c(0.10, 0.10),
                             psi.delta=0,
                             method.de.gene="t.test",
                             method.adjust.de.gene="fdr",
                             downsample=FALSE,
                             show.progress=FALSE,
                             level="gene.spliced"
                             )

head(marvel.demo$DE$Exp.Spliced$Table)
##              gene_id gene_short_name      gene_type n.cells.g1 n.cells.g2
## 1 ENSG00000128739.22           SNRPN protein_coding         15         15
## 2 ENSG00000067225.18             PKM protein_coding         15         15
## 3 ENSG00000137309.19           HMGA1 protein_coding         15         15
## 4  ENSG00000239672.7            NME1 protein_coding         15         13
## 5  ENSG00000120675.6         DNAJC15 protein_coding         14         15
## 6 ENSG00000154473.18            BUB3 protein_coding         15         14
##    mean.g1   mean.g2     log2fc statistic        p.val    p.val.adj
## 1 9.203101  8.331827 -0.8712731  6.773031 9.876425e-07 3.851806e-05
## 2 7.896393 10.207866  2.3114738 -6.477287 2.845515e-06 5.548755e-05
## 3 7.276139  6.060553 -1.2155857  6.148961 5.694655e-06 7.403052e-05
## 4 8.985144  5.082584 -3.9025599  5.627087 5.476249e-05 4.934664e-04
## 5 3.871605  6.065549  2.1939435 -4.706610 6.484850e-05 4.934664e-04
## 6 7.893652  4.895821 -2.9978306  5.221021 7.591791e-05 4.934664e-04

Volcano plot: Spliced genes

# Plot: No annotation
marvel.demo <- PlotDEValues(MarvelObject=marvel.demo,
                            method=c("ad", "dts"),
                            psi.pval=c(0.10, 0.10),
                            psi.delta=0,
                            gene.pval=0.10,
                            gene.log2fc=0.5,
                            point.size=0.1,
                            xlabel.size=8,
                            level="gene.spliced",
                            anno=FALSE
                            )

marvel.demo$DE$Exp.Spliced$Plot

plot of chunk unnamed-chunk-37

marvel.demo$DE$Exp.Spliced$Summary
##    sig freq
## 1   up    4
## 2 down   10
## 3 n.s.   25
# Plot: Annotate top genes
results <- marvel.demo$DE$Exp.Spliced$Table

index <- which((results$log2fc > 2 | results$log2fc < -2) & -log10(results$p.val.adj) > 1)
gene_short_names <- results[index, "gene_short_name"]

marvel.demo <- PlotDEValues(MarvelObject=marvel.demo,
                            method=c("ad", "dts"),
                            psi.pval=c(0.10, 0.10),
                            psi.delta=0,
                            gene.pval=0.10,
                            gene.log2fc=0.5,
                            point.size=0.1,
                            xlabel.size=8,
                            level="gene.spliced",
                            anno=TRUE,
                            anno.gene_short_name=gene_short_names
                            )

marvel.demo$DE$Exp.Spliced$Plot

plot of chunk unnamed-chunk-38

Principal component analysis

| Dimension reduction analysis such as principal component analysis (PCA) enables us to investigate if phenotypically different cell populations are transcriptomically distinct from one another. | This may be done in a supervised or unsupervised manner. The former approach uses all expressed genes or splicing events while the latter approach uses pre-determined features, such as genes and splicing event obtained from differential expression analysis. | Here, we will assess if splicing represents an additional layer of heterogeneity underlying gene expression profile. We will also demonstrate how to retrieve differentially expressed genes and differentially spliced genes from the DE analysis outputs to be used as features in PCA.

DE genes

# Define sample groups
    # Retrieve sample metadata
    df.pheno <- marvel.demo$SplicePheno

    # Group 1
    sample.ids.1 <- df.pheno[which(df.pheno$cell.type=="iPSC"), "sample.id"]

    # Group 2
    sample.ids.2 <- df.pheno[which(df.pheno$cell.type=="Endoderm"), "sample.id"]

    # Merge
    cell.group.list <- list("iPSC"=sample.ids.1,
                            "Endoderm"=sample.ids.2
                            )

# Retrieve DE genes
  # Retrieve DE result table
  results.de.exp <- marvel.demo$DE$Exp$Table    

  # Retrieve relevant gene_ids
  index <- which(results.de.exp$p.val.adj < 0.10 & abs(results.de.exp$log2fc) > 0.5)
  gene_ids <- results.de.exp[index, "gene_id"]

# Reduce dimension
marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=gene_ids,
                      point.size=2.5,
                      level="gene"
                      )

marvel.demo$PCA$Exp$Plot

plot of chunk unnamed-chunk-39

DE splicing

# Retrieve DE tran_ids
method <- c("ad", "dts")

tran_ids.list <- list()

for(i in 1:length(method)) {

    results.de.psi <- marvel.demo$DE$PSI$Table[[method[i]]]
    index <- which(results.de.psi$p.val.adj < 0.10 & results.de.psi$outlier==FALSE)
    tran_ids <- results.de.psi[index, "tran_id"]
    tran_ids.list[[i]] <- tran_ids

}

tran_ids <- unique(unlist(tran_ids.list))

# Reduce dimension
marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

marvel.demo$PCA$PSI$Plot

plot of chunk unnamed-chunk-40

non-DE genes

# Retrieve relevant gene_ids
results.de.exp <- marvel.demo$DE$Exp$Table
index <- which(results.de.exp$p.val.adj < 0.10 & abs(results.de.exp$log2fc) > 0.5)
gene_ids <- results.de.exp[-index, "gene_id"]

# Reduce dimension
marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=gene_ids,
                      point.size=2.5,
                      level="gene"
                      )

marvel.demo$PCA$Exp$Plot

plot of chunk unnamed-chunk-41

Splicing (non-DE genes)

# Retrieve non-DE gene_ids
results.de.exp <- marvel.demo$DE$Exp$Table
index <- which(results.de.exp$p.val.adj > 0.10 )
gene_ids <- results.de.exp[, "gene_id"]

# Retrieve tran_ids
df.feature <- do.call(rbind.data.frame, marvel.demo$SpliceFeatureValidated)
df.feature <- df.feature[which(df.feature$gene_id %in% gene_ids), ]

# Reduce dimension: All DE splicing events
tran_ids <- df.feature$tran_id

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.all <- marvel.demo$PCA$PSI$Plot

# Reduce dimension: SE
tran_ids <- df.feature[which(df.feature$event_type=="SE"), "tran_id"]

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.se <- marvel.demo$PCA$PSI$Plot

# Reduce dimension: MXE
tran_ids <- df.feature[which(df.feature$event_type=="MXE"), "tran_id"]

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.mxe <- marvel.demo$PCA$PSI$Plot

# Reduce dimension: RI
tran_ids <- df.feature[which(df.feature$event_type=="RI"), "tran_id"]

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.ri <- marvel.demo$PCA$PSI$Plot

# Reduce dimension: A5SS
tran_ids <- df.feature[which(df.feature$event_type=="A5SS"), "tran_id"]

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.a5ss <- marvel.demo$PCA$PSI$Plot

# Reduce dimension: A3SS
tran_ids <- df.feature[which(df.feature$event_type=="A3SS"), "tran_id"]

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.a3ss <- marvel.demo$PCA$PSI$Plot

# Reduce dimension: AFE
tran_ids <- df.feature[which(df.feature$event_type=="AFE"), "tran_id"]

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.afe <- marvel.demo$PCA$PSI$Plot

# Reduce dimension: 
tran_ids <- df.feature[which(df.feature$event_type=="ALE"), "tran_id"]

marvel.demo <- RunPCA(MarvelObject=marvel.demo,
                      cell.group.column="cell.type",
                      cell.group.order=c("iPSC", "Endoderm"),
                      cell.group.colors=NULL,
                      min.cells=5,
                      features=tran_ids,
                      point.size=2.5,
                      level="splicing",
                      method.impute="random",
                      seed=1
                      )

plot.ale <- marvel.demo$PCA$PSI$Plot

# Arrange and view plots
# Read plots from right to left for each row
grid.arrange(plot.all, plot.se, 
             plot.mxe, plot.ri, 
             plot.a5ss, plot.a3ss, 
             plot.afe, plot.ale,
             nrow=4)

plot of chunk unnamed-chunk-42

Modality dynamics

| Modality dynamics reveals the change in splicing pattern (modality) from one cell population (iPSCs) to another (endoderm cells). The modality dynamics from one cell population to another can be classified into three categories, namely explicit, implicit, and restricted.

| Here, we will perform modality dynamics analysis among differentially spliced events. Representative examples for each modality dynamics classification will also be shown. This section also introduces our ad hoc plot function PlotValues for plotting selected splicing events.

Assign dynamics

# Define sample groups
    # Retrieve sample metadata
    df.pheno <- marvel.demo$SplicePheno

    # Group 1
    sample.ids.1 <- df.pheno[which(df.pheno$cell.type=="iPSC"), "sample.id"]

    # Group 2
    sample.ids.2 <- df.pheno[which(df.pheno$cell.type=="Endoderm"), "sample.id"]

    # Merge
    cell.group.list <- list("iPSC"=sample.ids.1,
                            "Endoderm"=sample.ids.2
                            )

# Assign modality dynamics
marvel.demo <- ModalityChange(MarvelObject=marvel.demo,
                       method=c("ad", "dts"),
                       psi.pval=c(0.10, 0.10)
                       )

marvel.demo$DE$Modality$Plot

plot of chunk unnamed-chunk-43

head(marvel.demo$DE$Modality$Table)
##                                                                       tran_id
## 1                       chr17:8383254:8382781|8383157:-@chr17:8382143:8382315
## 2               chr17:8383157:8383193|8382781:8383164:-@chr17:8382143:8382315
## 3 chr15:24962114:24962209:+@chr15:24967029:24967152:+@chr15:24967932:24968082
## 4               chr8:144792587:144792245|144792366:-@chr8:144791992:144792140
## 5     chr8:144792366:144792423|144792245:144792389:-@chr8:144791992:144792140
## 6     chr5:150449703:150449739|150449492:150449696:-@chr5:150447585:150447735
##   event_type            gene_id gene_short_name      gene_type
## 1       A5SS ENSG00000161970.15           RPL26 protein_coding
## 2        AFE ENSG00000161970.15           RPL26 protein_coding
## 3         SE ENSG00000128739.22           SNRPN protein_coding
## 4       A5SS ENSG00000161016.17            RPL8 protein_coding
## 5        AFE ENSG00000161016.17            RPL8 protein_coding
## 6        AFE ENSG00000164587.13           RPS14 protein_coding
##   modality.bimodal.adj.g1 modality.bimodal.adj.g2 modality.change
## 1      Excluded.Dispersed        Excluded.Primary        Implicit
## 2      Excluded.Dispersed        Excluded.Primary        Implicit
## 3      Excluded.Dispersed        Excluded.Primary        Implicit
## 4      Excluded.Dispersed        Excluded.Primary        Implicit
## 5      Excluded.Dispersed        Excluded.Primary        Implicit
## 6        Excluded.Primary        Excluded.Primary      Restricted
marvel.demo$DE$Modality$Plot.Stats
##   modality.change freq      pct
## 1        Explicit    8 15.38462
## 2        Implicit   22 42.30769
## 3      Restricted   22 42.30769

Explicit

# Example 1
tran_id <- "chr4:108620569:108620600|108620656:108620712:+@chr4:108621951:108622024"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.1 <- marvel.demo$adhocPlot$PSI

# Example 2
tran_id <- "chr12:110502049:110502117:-@chr12:110499535:110499546:-@chr12:110496012:110496203"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.2 <- marvel.demo$adhocPlot$PSI

# Example 3
tran_id <- "chr9:35685269:35685339:-@chr9:35685064:35685139:-@chr9:35684732:35684807:-@chr9:35684488:35684550"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.3 <- marvel.demo$adhocPlot$PSI

# Example 4
tran_id <- "chr11:85981129:85981228:-@chr11:85978070:85978093:-@chr11:85976623:85976682"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.4 <- marvel.demo$adhocPlot$PSI

# Arrange and view plots
# Read plots from right to left for each row
grid.arrange(plot.1, plot.2, 
             plot.3, plot.4,
             nrow=1)

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Implicit

# Example 1
tran_id <- "chr17:8383254:8382781|8383157:-@chr17:8382143:8382315"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.1 <- marvel.demo$adhocPlot$PSI

# Example 2
tran_id <- "chr17:8383157:8383193|8382781:8383164:-@chr17:8382143:8382315"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.2 <- marvel.demo$adhocPlot$PSI

# Example 3
tran_id <- "chr15:24962114:24962209:+@chr15:24967029:24967152:+@chr15:24967932:24968082"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.3 <- marvel.demo$adhocPlot$PSI

# Example 4
tran_id <- "chr8:144792587:144792245|144792366:-@chr8:144791992:144792140"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.4 <- marvel.demo$adhocPlot$PSI

# Arrange and view plots
# Read plots from right to left for each row
grid.arrange(plot.1, plot.2, 
             plot.3, plot.4,
             nrow=1)

plot of chunk unnamed-chunk-45

Restricted

# Example 1
tran_id <- "chr5:150449703:150449739|150449492:150449696:-@chr5:150447585:150447735"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.1 <- marvel.demo$adhocPlot$PSI

# Example 2
tran_id <- "chr12:56725340:56724962|56725263:-@chr12:56724452:56724523"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.2 <- marvel.demo$adhocPlot$PSI

# Example 3
tran_id <- "chr10:78037194:78037304:+@chr10:78037439:78037441:+@chr10:78040204:78040225"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.3 <- marvel.demo$adhocPlot$PSI

# Example 4
tran_id <- "chr10:78037194:78037304:+@chr10:78037439|78040204:78040225"

marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                          cell.group.list=cell.group.list,
                          feature=tran_id,
                          xlabels.size=5,
                          level="splicing",
                          min.cells=5
                          )

plot.4 <- marvel.demo$adhocPlot$PSI

# Arrange and view plots
# Read plots from right to left for each row
grid.arrange(plot.1, plot.2, 
             plot.3, plot.4,
             nrow=1)

plot of chunk unnamed-chunk-46

Gene-splicing dynamics

| MARVEL's integrated differential gene and splicing analysis enables us to investigate how gene expression changes relative to splicing changes when iPSCs differentiate into endoderm cells. The gene-splicing dynamics may be classified into four categories, namely coordinated, opposing, isoform-switching, and complex.

| Here, we will explore the gene-splicing dynamics of genes that are differentially spliced between iPSCs and endoderm cells. Representative examples of each dynamic will also be shown. This section also utilises the ad hoc plotting function PlotValues for plotting selected splicing events and genes. | Please note that the function CompareValues with the level option set to gene.spliced needs to be executed prior to proceeding with gene-splicing dynamics analysis below. Kindly refer to Differential (spliced) gene analysis section of this tutorial.

Assign dynamics

marvel.demo <- IsoSwitch(MarvelObject=marvel.demo,
                         method=c("ad", "dts"),
                         psi.pval=c(0.10, 0.10),
                         psi.delta=0,
                         gene.pval=0.10,
                         gene.log2fc=0.5
                         )

marvel.demo$DE$Cor$Plot

plot of chunk unnamed-chunk-47

head(marvel.demo$DE$Cor$Table)
##               gene_id gene_short_name      gene_type         cor
## 3  ENSG00000128739.22           SNRPN protein_coding Coordinated
## 7  ENSG00000196531.10            NACA protein_coding  Iso-Switch
## 17  ENSG00000239672.7            NME1 protein_coding Coordinated
## 19 ENSG00000109475.16           RPL34 protein_coding  Iso-Switch
## 20 ENSG00000156482.11           RPL30 protein_coding  Iso-Switch
## 21 ENSG00000111237.18           VPS29 protein_coding  Iso-Switch
marvel.demo$DE$Cor$Plot.Stats
##           cor freq      pct
## 1 Coordinated    8 20.51282
## 2    Opposing    6 15.38462
## 3  Iso-Switch   25 64.10256

Coordinated

# Define cell groups
    # Retrieve sample metadata
    df.pheno <- marvel.demo$SplicePheno

    # Group 1
    sample.ids.1 <- df.pheno[which(df.pheno$cell.type=="iPSC"), "sample.id"]

    # Group 2
    sample.ids.2 <- df.pheno[which(df.pheno$cell.type=="Endoderm"), "sample.id"]

    # Merge
    cell.group.list <- list("iPSC"=sample.ids.1,
                            "Endoderm"=sample.ids.2
                            )

# Example 1
  # Gene
  df.feature <- marvel.demo$GeneFeature
  gene_id <- df.feature[which(df.feature$gene_short_name=="CMC2"), "gene_id"]

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=gene_id,
                            maintitle="gene_short_name",
                            xlabels.size=7,
                            level="gene"
                            )

  plot.1_gene <- marvel.demo$adhocPlot$Exp

  # Splicing
  tran_id <- "chr16:80981806:80981877:-@chr16:80980808:80980879|80976003:80976179"

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=tran_id,
                            xlabels.size=7,
                            level="splicing",
                            min.cells=5
                            )

  plot.1_splicing <- marvel.demo$adhocPlot$PSI

# Example 2
  # Gene
  df.feature <- marvel.demo$GeneFeature
  gene_id <- df.feature[which(df.feature$gene_short_name=="HNRNPC"), "gene_id"]

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=gene_id,
                            maintitle="gene_short_name",
                            xlabels.size=7,
                            level="gene"
                            )

  plot.2_gene <- marvel.demo$adhocPlot$Exp

  # Splicing
  tran_id <- "chr14:21231072:21230958|21230997:-@chr14:21230319:21230366"

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=tran_id,
                            xlabels.size=7,
                            level="splicing",
                            min.cells=5
                           )

  plot.2_splicing <- marvel.demo$adhocPlot$PSI

# Arrange and view plots
# Read plots from right to left for each row
grid.arrange(plot.1_gene, plot.1_splicing, 
             plot.2_gene, plot.2_splicing,
             nrow=2)

plot of chunk unnamed-chunk-48

Opposing

# Example 1
  # Gene
  df.feature <- marvel.demo$GeneFeature
  gene_id <- df.feature[which(df.feature$gene_short_name=="APOO"), "gene_id"]

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=gene_id,
                            maintitle="gene_short_name",
                            xlabels.size=7,
                            level="gene"
                            )

  plot.1_gene <- marvel.demo$adhocPlot$Exp

  # Splicing
  tran_id <- "chrX:23840313:23840377:-@chrX:23833353:23833612|23833367:23833510"

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=tran_id,
                            xlabels.size=7,
                            level="splicing",
                            min.cells=5
                            )

  plot.1_splicing <- marvel.demo$adhocPlot$PSI

# Example 2
  # Gene
  df.feature <- marvel.demo$GeneFeature
  gene_id <- df.feature[which(df.feature$gene_short_name=="BUB3"), "gene_id"]

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=gene_id,
                            maintitle="gene_short_name",
                            xlabels.size=7,
                            level="gene"
                            )

  plot.2_gene <- marvel.demo$adhocPlot$Exp

  # Splicing
  tran_id <- "chr10:123162612:123162828:+@chr10:123163820:123170467|123165047:123165365"

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=tran_id,
                            xlabels.size=7,
                            level="splicing",
                            min.cells=5
                            )

  plot.2_splicing <- marvel.demo$adhocPlot$PSI

# Arrange and view plots
# Read plots from right to left for each row
grid.arrange(plot.1_gene, plot.1_splicing, 
             plot.2_gene, plot.2_splicing,
             nrow=2)

plot of chunk unnamed-chunk-49

Iso-switch

# Example 1
  # Gene
  df.feature <- marvel.demo$GeneFeature
  gene_id <- df.feature[which(df.feature$gene_short_name=="AC004086.1"), "gene_id"]

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=gene_id,
                            maintitle="gene_short_name",
                            xlabels.size=7,
                            level="gene"
                            )

  plot.1_gene <- marvel.demo$adhocPlot$Exp

  # Splicing
  tran_id <- "chr12:112409641:112409411|112409587:-@chr12:112408420:112408656"

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=tran_id,
                            xlabels.size=7,
                            level="splicing",
                            min.cells=5
                            )

  plot.1_splicing <- marvel.demo$adhocPlot$PSI

# Example 2
  # Gene
  df.feature <- marvel.demo$GeneFeature
  gene_id <- df.feature[which(df.feature$gene_short_name=="ACP1"), "gene_id"]

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=gene_id,
                            maintitle="gene_short_name",
                            xlabels.size=7,
                            level="gene"
                            )

  plot.2_gene <- marvel.demo$adhocPlot$Exp

  # Splicing
  tran_id <- "chr2:271866:271939:+@chr2:272037:272150:+@chr2:272192:272305:+@chr2:275140:275201"

  marvel.demo <- PlotValues(MarvelObject=marvel.demo,
                            cell.group.list=cell.group.list,
                            feature=tran_id,
                            xlabels.size=7,
                            level="splicing",
                            min.cells=5
                            )

  plot.2_splicing <- marvel.demo$adhocPlot$PSI

# Arrange and view plots
# Read plots from right to left for each row
grid.arrange(plot.1_gene, plot.1_splicing, 
             plot.2_gene, plot.2_splicing,
             nrow=2)

plot of chunk unnamed-chunk-50

Gene ontology analysis

| Gene ontology analysis or pathway enrichment analysis categorises the differentially spliced genes between iPSCs and endoderm cell into biological pathways. This may identify sets of genes with similar function or belong to similar biological pathways that are concurrently spliced. | Gene ontology analysis represents one of the two functional annotation features of MARVEL. The other functional annotation feature is nonsense-mediated (NMD) analysis.

marvel.demo <- BioPathways(MarvelObject=marvel.demo,
                           method=c("ad", "dts"),
                           pval=0.10,
                           species="human"
                           )

head(marvel.demo$DE$BioPathways$Table)
# Plot top pathways
df <- marvel.demo$DE$BioPathways$Table
go.terms <- df$Description[c(1:10)]

marvel.demo <- BioPathways.Plot(MarvelObject=marvel.demo,
                                go.terms=go.terms,
                                y.label.size=10
                                )

marvel.demo$DE$BioPathways$Plot

plot of chunk unnamed-chunk-52

Companion tool: VALERIE

| From this tutorial, we identified over 1,000 differentially spliced events. We would like to introduce VALERIE (Visulazing ALternative splicing Events from RIbonucleic acid Experiments) - a visualisation platform for visualising alternative splicing events at single-cell resolution. | The tutorial for using VALERIE for investigating these differentially spliced events can be found here: https://wenweixiong.github.io/VALERIE. The R package may be installed from Github here: https://github.com/wenweixiong/VALERIE.

These binaries (installable software) and packages are in development.
They may not be fully stable and should be used with caution. We make no claims about them.