RNAseq Pipelines – Lab Meeting

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RNAseq Pipelines – Lab Meeting

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RNAseq Pipelines

Lab Meeting

Emilio Palumbo June 25, 2014

Blueprint pipeline

Blueprint pipeline

RNAseq pipeline written in Bash
  • mapping (GEMtools)
  • bigwig
  • contig
  • quantification (Flux Capacitor)
  • BAM statistics (RSeQC)

Bash

#!/bin/bash
  • widely supported on any Linux platform
  • most bioinformaticians are used to it

SGE options

# Force bash shell
#$ -S /bin/bash
#
# Export environment variables
#$ -V
#
# Use current working directory as job working directory
#$ -cwd
#
# Rename stdout and stderr files
#$ -o $JOB_NAME.out
#$ -e $JOB_NAME.err

Queue, memory and time must be specified when submitting the job.

Modular execution

# getting pipeline steps to be executed
steps=(mapping bigwig contig flux)

if [[ "$@" ]]; then
    read -ra steps <<< "$@"
fi

for step in ${steps[@]}; do
    case $step in
        mapping)
            doMapping="true"
            ;;
        bigwig)
            doBigWig="true"
            ;;
        contig)
            doContig="true"
            ;;
        quant|flux)
            doFlux="true"
            ;;
    esac
done

Run specific steps: 

blueprint.pipeline.sh ... -- contig flux

Monolitic pipeline

  • pipeline steps are executed sequencially
  • one cluster node per run is used weak parallelization
  • bad resource management - not all steps use the same amount of cpus/memory

Batch execution

Some extra code required: 

#!/bin/bash

base="/users/rg/epalumbo/projects/BluePrint"
threads="8"
vmem="64G"
fluxMem=$((${vmem:0:$((${#vmem}-1))}/2))${vmem:$((${#vmem}-1)):1}
rtime="72:00:00"
do=${1-"echo"}

while read lab id path strand sex; do
    quality="33"
    genome="$base/genome/Homo_sapiens.GRCh37.chromosomes.chr.fa"
    annotation="$base/annotation/gencode.v15.annotation.gtf"
    if [[ $sex == 'Female' ]]; then
        genome="$base/genome/Homo_sapiens.GRCh37.chromosomes.female.chr.fa"
        annotation="$base/annotation/gencode.v15.annotation.female.gtf"
    fi
    sample=`echo $id | cut -d"_" -f1`
    readGroup="ID=$id,PL=ILLUMINA,PU=$id,LB=Blueprint,SM=$sample,DT=`date +"%Y-%m-%dT%H:%M:%S%z"`,CN=$lab,DS=Blueprint"
    command="qsub -pe smp $threads -l virtual_free=$vmem,h_rt=$rtime -q rg-el6,long -N $id.pipeline"
    command=$command" ../blueprint.pipeline.sh -i `basename $path` -g $genome -a $annotation -q $quality -t $threads --paired --read-group $readGroup -r 150 --read-strand ${strand^^} --flux-mem $fluxMem"

    if [ ! -d $id ]; then
        $do "mkdir $id"
        $do "ln -s $path $id"
        $do "ln -s ${path//_1/_2} $id"
    fi
    $do "cd $id"
    $do "$command"
    $do "cd ..""
done

Data management

User has to keep metadata and file information:

  • tsv/csv files
  • Google spreadsheets
  • Index files

Idxtools

github.com/emi80/idxtools

Index files

Plain text database files to store metadata information for files and their content

Format

<filepath>TAB<attributes_list>

with attribute_list as a semicolon separated list of key=value strings:

/path/to/file    size=100; id=1; class=MyClass; type=MyType

Idxtools

Python API and commandline tool to create, query and modify index files

Dataset

Dataset is the main data object

Metadata: 

.   id=test1; quality=33; sex=female;

Files: 

/data/test1_1.fastq.gz id=test1; quality=33; sex=female; type=fastq; view=FqRd1;
/data/test1.bam id=test1; quality=33; sex=female; type=bam; view=Alignments;

Format.json

Specifies syntax and vocabulary of an index file
{
  "colsep": "\t",
  "id": "labExpId",
  "kw_sep": " ",
  "sep": "=",
  "trail": ";",
  "fileinfo": [
    "path",
    "size",
    "md5",
    "type",
    "view"
  ]
}

Mapping attribute keys

Useful when importing from csv/tsv: 

{
  "colsep": "\t",
  "sep": "=",
  "trail": ";",
  "kw_sep": " ",
  "id": "labExpId",
  "fileinfo": [
    "path",
    "size",
    "md5",
    "type",
    "view",
    "submittedDataVersion"
  ],
  "map": {
    "BASE_COUNT": "baseCount",
    "BIOMATERIAL_PROVIDER": "",
    "BIOMATERIAL_TYPE": "localization",
    "CELL_TYPE": "cell",
    "CENTER_NAME": "lab",
    "DISEASE": "",
    "DONOR_AGE": "age",
    "DONOR_ETHNICITY": "ethnicity",
    "DONOR_HEALTH_STATUS": "",
    "DONOR_ID": "donorId",
    "DONOR_REGION_OF_RESIDENCE": "",
    "DONOR_SEX": "sex",
    "EXPERIMENT_ID": "",
    "EXPERIMENT_TYPE": "",
    "_FILE": "path",
    "FILE_MD5": "md5",
    "FILE_SIZE": "size",
    "FIRST_SUBMISSION_DATE": "dateSubmittedFirst",
    "INSTRUMENT_MODEL": "",
    "INSTRUMENT_PLATFORM": "seqPlatform",
    "LIBRARY_LAYOUT": "",
    "LIBRARY_NAME": "libProtocol",
    "LIBRARY_STRATEGY": "dataType",
    "MOLECULE": "rnaExtract",
    "READ_COUNT": "readCount",
    "READ_TYPE": "readType",
    "READ_QUALITIES": "quality",
    "READ_STRAND": "readStrand",
    "RUN_ID": "labExpId",
    "RUN_NAME": "",
    "SAMPLE_ID": "sraSampleAccession",
    "SAMPLE_NAME": "labProtocolId",
    "SEQ_RUNS_COUNT": "seqRun",
    "SPECIMEN_PROCESSING": "",
    "SPECIMEN_STORAGE": "",
    "STUDY_ID": "sraStudyAccession",
    "STUDY_NAME": "",
    "SUBMISSION_DATE": "dateSubmitted",
    "SUBMISSION_ID": "",
    "TISSUE": "tissue",
    "TWIN_PAIR_ID": "",
    "TYPE": "type",
    "WITHDRAWN": ""
  }
}

Specifying input files

Index and format files can be specified as command line options or as environment variables 
# command line
$ idxtools -i index.txt -f format.json [command]

# environment variables
export IDX_FILE=/path/to/index.txt
export IDX_FORMAT=/path/to/format.json

$ idxtools show

Using absolute paths to make the index available from any folder

Importing TSV/CSV

A CSV file, test.csv:

path,labExpId,quality,sex,type,view
/data/test1_1.fastq.gz,test1,33,female,fastq,FqRd1
/data/test1_2.fastq.gz,test1,33,female,fastq,FqRd2
/data/test2_1.fastq.gz,test2,64,male,fastq,FqRd1
/data/test2_2.fastq.gz,test2,64,male,fastq,FqRd2
/data/test2.bam,test2,64,male,bam,Alignments

# import csv and show
$ export IDX_FORMAT=$PWD/format.json
$ idxtools -i test.csv show
/data/test1_1.fastq.gz labExpId=test1; type=fastq; view=FqRd1; quality=33; sex=female;
/data/test1_2.fastq.gz labExpId=test1; type=fastq; view=FqRd2; quality=33; sex=female;
/data/test2_1.fastq.gz labExpId=test2; type=fastq; view=FqRd1; quality=64; sex=male;
/data/test2_2.fastq.gz labExpId=test2; type=fastq; view=FqRd2; quality=64; sex=male;
/data/test2.bam    labExpId=test2; type=bam; view=Alignments; quality=64; sex=male;

# import csv and save to file
$ idxtools -i test.csv -f format.json show -o index.txt
$ export IDX_FILE=$PWD/index.txt

Querying the index

# query by attributes
$ idxtools show id=test2
/data/test2_1.fastq.gz labExpId=test2; type=fastq; view=FqRd1; quality=64; sex=male;
/data/test2_2.fastq.gz labExpId=test2; type=fastq; view=FqRd2; quality=64; sex=male;
/data/test2.bam    labExpId=test2; type=bam; view=Alignments; quality=64; sex=male;

$ idxtools show view=FqRd1
./data/test1_1.fastq.gz labExpId=test1; type=fastq; view=FqRd1; quality=33; sex=female;
./data/test2_1.fastq.gz labExpId=test2; type=fastq; view=FqRd1; quality=64; sex=male;

$ idxtools show type=bam
./data/test2.bam    labExpId=test2; type=bam; view=Alignments; quality=64; sex=male;

Querying the index

# queries use regular expressions
# query by id starting with t
$ idxtools show id=t # equivalent to: idxtools show id="^t"
/data/test1_1.fastq.gz labExpId=test1; type=fastq; view=FqRd1; quality=33; sex=female;
/data/test1_2.fastq.gz labExpId=test1; type=fastq; view=FqRd2; quality=33; sex=female;
/data/test2_1.fastq.gz labExpId=test2; type=fastq; view=FqRd1; quality=64; sex=male;
/data/test2_2.fastq.gz labExpId=test2; type=fastq; view=FqRd2; quality=64; sex=male;
/data/test2.bam    labExpId=test2; type=bam; view=Alignments; quality=64; sex=male;

# exact queries
$ idxtools show id=t --exact

# operations on numeric attributes
$ idxtools show quality=">40"
/data/test2_1.fastq.gz labExpId=test2; type=fastq; view=FqRd1; quality=64; sex=male;
/data/test2_2.fastq.gz labExpId=test2; type=fastq; view=FqRd2; quality=64; sex=male;
/data/test2.bam    labExpId=test2; type=bam; view=Alignments; quality=64; sex=male;
$ idxtools show quality="<40"
/data/test1_1.fastq.gz labExpId=test1; type=fastq; view=FqRd1; quality=33; sex=female;
/data/test1_2.fastq.gz labExpId=test1; type=fastq; view=FqRd2; quality=33; sex=female;

Query output

The default output format for queries is the index file format but it can be changed to TSV with the -t option
# TSV output - single attribute
$ idxtools show view=FqRd1 -t path
/data/test1_1.fastq.gz
/data/test2_1.fastq.gz

# TSV output - multiple attributes
$ idxtools show view=FqRd1 -t id,path
test1   ./data/test1_1.fastq.gz
test2   ./data/test2_1.fastq.gz

# with header
$ idxtools show view=FqRd1 -t id,path --header
id  path
test1   ./data/test1_1.fastq.gz
test2   ./data/test2_1.fastq.gz

attribute names comma separated list with NO space

Adding files

Files can be easily added to the index
# add /data/test1.bam
$ idxtools add path=/data/test1.bam id=test1 type=bam view=Alignments

# check
$ idxtools show type=bam
/data/test1.bam    labExpId=test1; type=bam; view=Alignments; quality=33; sex=female;
/data/test2.bam    labExpId=test2; type=bam; view=Alignments; quality=64; sex=male;

Removing files

Files can be easily removed from the index
# remove /data/test1.bam
$ idxtools remove /data/test1.bam

# check
$ idxtools show type=bam
/data/test2.bam    labExpId=test2; type=bam; view=Alignments; quality=64; sex=male;

Removing datasets

Datasets can also be removed from the index
# remove test2
$ idxtools remove id=test2

# check
$ idxtools show
./data/test1_1.fastq.gz labExpId=test1; type=fastq; view=FqRd1; quality=33; sex=female;
./data/test1_2.fastq.gz labExpId=test1; type=fastq; view=FqRd2; quality=33; sex=female;

JIP

pyjip.readthedocs.org

JIP

A pipeline system that helps to manage large numbers of jobs on a compute cluster
  • dependency support
  • automatic expansions
  • simplified management of jobs and resources
  • SQlite database for Jobs data

Tools

The smallest unit of execution in JIP is a tool.

Tools can be implemented in various ways:

  • JIP scritps
  • Python APIs
Tools can be combined to pipelines to build bigger workflows.

Configuration

{
    "cluster": "jip.cluster.SGE",
    "sge" : {
        "threads_pe": "smp",
        "time_limit": "h_rt"
    },
    "profiles": {
        "default": {
            "queue": "short,long,rg-el6",
            "time": "3h"
        },

        "long": {
            "queue": "long,rg-el6",
            "time": "24h",
            "mem": "64G"
        },
        "short": {
            "queue": "short",
            "time": "6h",
            "mem": "16G"
        }
    }
}

Run or submit commands

The jip bash command:

# run `hostname` locally
$ jip bash -c hostname

# submit
$ jip bash -s -c hostname

Check jobs with jip jobs:

# check the status of the job
$ jip jobs

Scripts

JIP scripts are extended Bash scripts: 

    #!/usr/bin/env jip
    # Greetings
    # usage:
    #     hello.jip <name>

    echo "Hello ${name}"

Make the file executable and you can use the JIP interpreter to run it or submit it: 

    $ chmod +x hello.jip
    # run the script
    $ ./hello.jip Joe
    # show dry run and command only
    $ ./hello.jip Joe -- --dry --show
    # submit the script run
    $ ./hello.jip Joe -- submit

Tools path

Scripts can be found automatically in your current folder or by setting the JIP_PATH environment variable. Use the jip tools command to list all the detected tools.
    $> export JIP_PATH=$PWD
    $> jip tools
    ...
            Name                                            Path
    ======================================================================================
    hello.jip                                 /users/rg/epalumbo/jip/hello.jip
    ...

Example tool

#!/bin/env jip
# Sort fastq file by read id
#
# Usage:
#   fastq-sort.jip [-i <fastq>] [-o <sorted_fastq>]
#
# Inputs:
#   -i, --input <input>   Input fastq [default: stdin]
#
# Outputs:
#   -o, --output <output>  Output sorted fastq [default: stdout]
#

${input|arg("cat ")|suf(" | ")}paste - - - - | sort -k1,1 | tr '\t' '\n' ${output|arg(">")}

Example tool with logic

#!/bin/env jip
# Run pigz on an input file
#
# Usage:
#   pigz.jip [-i <INPUT>] [-d] [-o <OUTPUT>]
#
# Inputs:
#   -i, --input <INPUT>  The input file [default: stdin]
#
# Outputs:
#   -o, --output <OUTPUT>  The output file [default: stdout]
#
# Options:
#   -d, --decompress  Decompress data

#%begin setup
add_option('stdout', False, hidden=False, short='-c')
if input.get():
    if opts['input'].raw().endswith('gz'):
        opts['decompress'] = True
    else:
        opts['decompress'] = False
if not output.get():
    opts['stdout'] = True
else:
    if output.get() == '${input}.gz' or output.get().replace('.gz','') == input.get():
        opts['output'].hidden = True
#%end

pigz -p ${JIP_THREADS} ${stdout|arg} ${decompress|arg} ${input|arg("")} ${output|arg(">")}

Example pipeline

#!/bin/env jip
#
# Sort and compress fastq
#
# Usage:
#   fq-pipe [-i <INPUT>] [-o <OUTPUT>]
#
# Inputs:
#   -i, --input <INPUT>  Input file [default: stdin]
#
# Outputs:
#   -o, --output <OUTPUT>  Output file [default: stdout]
#

#%begin pipeline
decomp_input = input
if type(input.raw()) == unicode and input.raw().endswith('gz'):
    decomp_input = job(temp=True).run('pigz', input=decomp_input)
sorted_fastq = job(temp=True).run('fastq-sort', input=decomp_input)
run('pigz', input=sorted_fastq, output=output)
#%end

Multiplexing

Automatic expansion of wildcard for multiple input files. The tool/pipeline will be replicated across all input files.
$ ./sort_fastq.jip -i *_1.fastq -- --dry --show
...
#####################
|  Job hierarchy    |
#####################
sort_fastq.0
sort_fastq.1
#####################
| Tasks:         2  |
| Jobs:          2  |
| Named Groups:  1  |
| Job Groups:    2  |
#####################

Job commands
============
### sort_fastq.0 -- Interpreter: bash
###   stdout: <default>
###   stderr: <default>
cat /home/epalumbo/testA_1.fastq | paste - - - - | sort -k1,1 | tr '\t' '\n' >/home/epalumbo/testA_1_sorted.fastq
###
### sort_fastq.1 -- Interpreter: bash
###   stdout: <default>
###   stderr: <default>
cat /home/epalumbo/testB_1.fastq | paste - - - - | sort -k1,1 | tr '\t' '\n' >/home/epalumbo/testB_1_sorted.fastq
###

Complex pipelines

SOLiD RNAseq pipeline

(shrimp + rsem + flux-capacitor) * 96:

  • index the genome
  • make the transcriptome and index it
  • convert csfasta to csfastq
  • genome mapping
  • transcriptome mapping
  • isoform quantifications
  • gene quantifications
concurrency problems submitting the jobs:

SQlite db limitation

Nextflow

Data-driven computational pipelines

www.nextflow.io

Nexflow

A fluent DSL modelled around the UNIX pipe concept, that simplifies writing parallel and scalable pipelines in a portable manner.
  • unified parallelism
  • continuous checkpoints
  • fast prototyping
  • reproducibility

Processes and channels

A Nextflow pipeline script is made by different isolated processes joined by asynchronous FIFO queues, called channels.
The pipeline execution flow is implicitly defined by the input and output channels declarations.

Configuration

Pipeline configuration properties are defined in a file named nextflow.config in the pipeline execution directory. 
// sample configuration
process {
    executor = 'sge'
    queue = 'rg-el6'
}

env {
    PATH="$PWD/gemtools-1.6.2-i3/bin:$PWD/flux-capacitor-1.2.4/bin:$PATH"
}

Scripts

Nextflow scripts are extended Groovy scripts: 

#!/usr/bin/env nextflow

str = Channel.from('hello', 'hola', 'bonjour', 'ciao')

process printHello {

   input:
   val str

   output:
   stdout into result

   """
   echo $str
   """
}

result.subscribe { print it }

Run a script

$ chmod +x hello.nf
$ ./hello.nf
N E X T F L O W  ~  version 0.8.2
[warm up] executor > local
[5a/d7e7d3] Submitted process > printHello (2)
[42/e78174] Submitted process > printHello (1)
[c3/88c277] Submitted process > printHello (4)
[7c/55887c] Submitted process > printHello (3)
bonjour
hola
ciao
hello

Example pipeline

#!/usr/bin/env nextflow

/*
 * Pipeline parameters that can be ovverridden by the command line parameter
 */
params.query = "$HOME/*.fa"
params.db = "$HOME/tools/blast-db/pdb/pdb"
params.out = "./result.txt"
params.chunkSize = 100

db = file(params.db)
fasta = Channel
            .fromPath(params.query)
            .splitFasta(by: params.chunkSize)

process blast {
    input:
    file 'query.fa' from fasta

    output:
    file 'top_hits'

    """
    blastp -db ${db} -query query.fa -outfmt 6 > blast_result
    cat blast_result | head -n 10 | cut -f 2 > top_hits
    """
}

process extract {
    input:
    file top_hits

    output:
    file 'sequences'

    "blastdbcmd -db ${db} -entry_batch top_hits | head -n 10 > sequences"
}

sequences
    .collectFile(name: params.out)
    .subscribe { println "Result saved at file: $it" }

Modules

Software environment managment

modules.sourceforge.net

Modules

Package for the dynamic modification of a user's environment via modulefiles.
  • loaded and unloaded dynamically and atomically
  • all popular shells supported
  • language binding for Perl and Python
  • managing different versions of applications
  • complete suites (metamodules)

Modules path

Paths of directories containing modulefiles are specified by setting the MODULEPATH environment variable.
$ echo $MODULEPATH
/software/rg/el6.3/modulefiles:/usr/share/Modules/modulefiles:/etc/modulefiles

# modulefile folder structure
$ tree /software/rg/el6.3/modulefiles
/software/rg/el6.3/modulefiles/
├── aspera
│   ├── 3.0.1
│   └── 3.3.3
├── bedtools
│   ├── 2.17.0
│   └── 2.19.1
...

Modulefiles

Contains the information needed to configure the shell for an application.

#%Module1.0
########################################################
#
# Author: Emilio Palumbo (emilio.palumbo@crg.es)
#
########################################################

set PROG_NAME       samtools
set PROG_VERSION    0.1.19
set PROG_HOME       /software/rg/el6.3/$PROG_NAME-$PROG_VERSION

proc ModulesHelp { } {

      puts stderr "$PROG_NAME version $PROG_VERSION"
}

module-whatis "loads the $PROG_NAME $PROG_VERSION module"

module-verbosity {on}

# Tests of consistency
# --------------------
# This application cannot be loaded if another $PROG_NAME modulefile was previously loaded

set namelow [string tolower $PROG_NAME]
conflict $namelow

### This shows info about loaded/unloaded module
if { [module-info mode] != "whatis" } {
   puts stderr "[module-info mode] [module-info name] (PATH, MANPATH, LD_LIBRARY_PATH, C_INCLUDE_PATH)"
}

prepend-path      PATH                 $PROG_HOME/bin
prepend-path      MANPATH              $PROG_HOME/man
prepend-path      LD_LIBRARY_PATH      $PROG_HOME/lib
prepend-path      C_INCLUDE_PATH       $PROG_HOME/include

Available modules

$ module avail
------------------------ /software/rg/el6.3/modulefiles ------------------------
aspera/3.0.1(default)              htslib/0.2.0-rc8(default)
aspera/3.3.3                       ipsa/1.0(default)
bedtools/2.17.0(default)           ipsa/1.1
bedtools/2.19.1                    jip-tools/1.0(default)
bowtie/1.0.1(default)              picard/1.81(default)
cufflinks/2.0.2                    pigz/2.2.5(default)
cufflinks/2.1.1                    pigz/2.3.1
cufflinks/2.2.1(default)           plink/1.07(default)
edirect/1.50(default)              python/2.7/2.7.3
emboss/6.6.0(default)              python/2.7/2.7.5
fastqc/0.10.1(default)             python/2.7/2.7.6(default)
flux/1.2.3                         python/2.7/2.7.6-sqlite
flux/1.2.4(default)                python/3/3.3.4
flux/1.2.4-SNAPSHOT                python-modules/2.6(default)
flux/1.3                           rsem/1.1.17
flux/1.4                           rsem/1.2.12(default)
flux/1.5.1                         samtools/0.1.18
flux/1.6                           samtools/0.1.19(default)
flux/1.6.1                         shrimp/2.2.3(default)
gemtools/1.6.1-i3                  sickle/1.210(default)
gemtools/1.6.2-i3(default)         sratoolkit/2.3.5(default)
gemtools/1.7.1-i3                  sublime-text/3-build-3059(default)
gemtools/1.7-i3                    texlive/2012(default)
glimmps                            ucsc/2013.02(default)
htop/1.0.2(default)                vcftools/0.2.12a(default)

------------------------ /usr/share/Modules/modulefiles ------------------------
dot         module-cvs  module-info modules     null        use.own

------------------------------- /etc/modulefiles -------------------------------
compat-openmpi-x86_64 openmpi-x86_64

Load/unload modules

# load default module version
$ module load samtools
load samtools/0.1.19 (PATH, MANPATH, LD_LIBRARY_PATH, C_INCLUDE_PATH)
$ which samtools
/software/rg/el6.3/samtools-0.1.19/bin/samtools

# load specific version
$ module load samtools/0.1.18
load samtools/0.1.18 (PATH, MANPATH, LD_LIBRARY_PATH, C_INCLUDE_PATH)
$ which samtools
/software/rg/el6.3/samtools-0.1.18/bin/samtools

# switch module version
$ module switch samtools samtools/0.1.19
switch1 samtools/0.1.18 (PATH, MANPATH, LD_LIBRARY_PATH, C_INCLUDE_PATH)
...
$ which samtools
/software/rg/el6.3/samtools-0.1.19/bin/samtools

Unload modules

# unload module
# equivalent to 'module unload samtools' but shorter!
$ module rm samtools
remove samtools/0.1.19 (PATH, MANPATH, LD_LIBRARY_PATH, C_INCLUDE_PATH)

# unload all loaded modules
$ module purge
remove samtools/0.1.19 (PATH, MANPATH, LD_LIBRARY_PATH, C_INCLUDE_PATH)
remove bedtools/2.17.0 (PATH)

List loaded modules

# list loaded modules
$ module list
Currently Loaded Modulefiles:
  1) samtools/0.1.19   2) bedtools/2.17.0

Grape 2

grape-pipeline.readthedocs.org

Current status

  • Beta version released
  • Idxtools data management
  • JIP execution engine
  • Embedded software management(grape-buildout)
  • Pipeline steps:
    • mappings
    • transcript quantifications

Work in progress

  • Nextflow execution engine
  • Modules/Docker software management
  • modularity (e.g. start from BAM file)