ikra v2.0.1 -RNAseq pipeline centered on Salmon-

A gene expression table (gene × sample) is automatically created from the experiment matrix. The output can be used as an input of idep. Ikra is an RNAseq pipeline centered on salmon.

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Note that sra-tools has to be installed locally. This is up to NCBI's tool upgrade. Please install sra-tools (>=2.10.7).

Usage

Usage: ikra.sh experiment_table.csv species \
        [--test, --fastq, --help, --without-docker, --udocker --protein-coding] \
        [--threads [VALUE]][--output [VALUE]]\
        [--suffix_PE_1 [VALUE]][--suffix_PE_2 [VALUE]]
  args
    1.experiment matrix(csv)
    2.reference(human or mouse)

Options:
  --test  test mode(MAX_SPOT_ID=100000).(dafault : False)
  --fastq use fastq files instead of SRRid. The extension must be foo.fastq.gz (default : False)
  -u, --udocker
  -w, --without-docker
  -pc, --protein-coding use protein coding transcripts instead of comprehensive transcripts. (default : True)
  -ct, --comprehensive-transcripts use comprehensive transcripts instead of protein coding transcripts. (default : False) 
  -t, --threads
  -o, --output  output file. (default : output.tsv)
  -l, --log  log file. (default : ikra.log)
  -a, --align carry out mapping onto a reference genome. hisat2 or star (default : None)
  -g, --gencode specify the version of gencode. (defalut : Mouse=26, Human=37)
  -s1, --suffix_PE_1    suffix for PE fastq files. (default : _1.fastq.gz)
  -s2, --suffix_PE_2    suffix for PE fastq files. (default : _2.fastq.gz)
  -h, --help    Show usage.
  -v, --version Show version.
  -r, --remove-intermediates Remove intermediate files

• test option limits the number of reads to 100,000 in each sample.
• udocker mode is for server environments that can only use User privileges. For more information https://github.com/indigo-dc/udocker.
• without-docker mode works with all tools installed. Not recommended.
• protein-coding mode restricts genes to protein coding genes only.
• threads
• output is output.tsv by default.
• align mode generates genome-mapped bam and bigwig files. Note that Salmon works quasi-alignment mode similarly as no-align mode. Generated bw files can be visualized on IGV or other genome browsers. experiment matrix should be separated by commas (csv format).

SRR mode

name	SRR	Layout	condition1 (optional)	...
Treg_LN_1	SRR5385247	SE	Treg	...
Treg_LN_2	SRR5385248	SE	Treg	...

fastq mode

name	fastq(PREFIX)	Layout	condition1 (optional)	...
Treg_LN_1	hoge/SRR5385247	SE	Treg	...
Treg_LN_2	hoge/SRR5385248	SE	Treg	...

• Denote names by connecting conditions and replicates with underscores. See idep's Naming convention in detail.
• The first three columns are required.
• If you want to use your own fastq file, add --fastq option. Ikra supports only .fq, .fq.gz, .fastq and fastq.gz.
• fastq file specifies path excluding fastq.gz or _1.fastq.gz and _2.fastq.gz. For example, hoge/SRR5385247.fastq.gz is described as hoge/SRR5385247.
• If suffix is not _1.fastq.gz or _2.fastq.gz, add -s1 and -s2 options.
• It is impossible for docker to specify a hierarchy above the execution directory, such as ../fq/**.fastq.gz, but it can be avoided by pasting a symbolic link. bonohu blog

Output

• output.tsv(scaledTPM)
• multiqc_report.html : including fastQC reports and mapping rate of salmon(mapping rate for transcripts)

output sample

	Treg_LN_1	Treg_LN_2
0610005C13Rik	0	0
0610006L08Rik	0	1
0610009B22Rik	4	10
...

Specification

• output is scaledTPM (see. Soneson, C., Love, M. I. & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research 4, 1521 (2015).)。
• —-gcBias option was added on salmon. You can refer to Mike Love's blog : RNA-seq fragment sequence bias.
• --validateMappings flag was also adopted. (You can’t use it while using alignment-base mode.) Please see salmon Frequently Asked Questions for further details.
• The reference for human is GENCODE Release 37 (GRCh38), and that for mouse is GENCODE Release M26 (GRCm38)

Major bugs that have fixed

tximport_R.R 2019/04/30

A serious bug was reported in the tximport_R.R and fixed. In the older version, Salmon's output and multiqc reports were correct and sometimes output.tsv were disturbed. Please update Ikra to the latest version. If you are using the old version(<1.1.1), please update and re-run ikra. We apologize for the inconvenience.

fasterq-dump error 2019/09/21

A bug has been reported that stops processing due to the following error in sra-tools. docker: Error response from daemon: OCI runtime create failed: container_linux.go:345: starting container process caused "exec: \"fasterq-dump\": executable file not found in $PATH": unknown. The latest version has already been corrected, so if you encounter the same error, please update to the latest version.

Install

All you need is git clone ikra, and install docker or udocker(v1.1.3). No need for installing plenty of softwares! If you don’t want to use docker (or udocker), you must install all softwares by yourself and use —-without-docker option.

$ git clone https://github.com/yyoshiaki/ikra.git

if you use SRR mode, install sra-toolkit locally.

Upgrade

$ git pull origin master

Version

 $ bash ikra.sh --version
 ...
 ikra v2.0.1 -RNAseq pipeline centered on Salmon-
 ...

Version of tools

• sra-tools : 2.10.9
• FastQC : 0.11.9
• MultiQC : 0.10.1
• Trim Galore! : 0.6.7
• Salmon : 1.4.0
• tximport : 1.6.0
• STAR : 2.7.8a
• Hisat2 : 2.2.1
• sambamba : 0.8.0
• deeptools : 3.5.1

Version of reference genome (when using alignment option)

• mouse：mm10 (GRCm38)
• human：hg38 (GRCh38)

Test

SE

SRR mode

$ cd test/Illumina_SE && bash ../../ikra.sh Illumina_SE_SRR.csv mouse --test -t 10

fastq mode

You can execute it after you execute SRR mode. (That is because you don’t have fastq files.)

$ cd test/Illumina_SE && bash ../../ikra.sh Illumina_SE_fastq.csv mouse --fastq -t 10

PE

SRR mode

$ cd test/Illumina_PE && bash ../../ikra.sh Illumina_PE_SRR.csv mouse --test -t 10

fastq mode

You can execute it after you execute SRR mode. (That is because you don’t have fastq files.)

$ cd test/Illumina_PE && bash ../../ikra.sh Illumina_PE_fastq.csv mouse --fastq -t 10

test all (for developers)

cd test && bash test.sh && bash test.full.sh

For Mac Users

Dr.Ota(DBCLS) solved the problem that salmon doesn’t work on Mac. The cause of the problem is that Docker is allocated only 2GB by default on Mac. The problem will be solved by allocating sufficient memory space(>=8Gb) for Docker, and applying and restarting Docker.

ikra pipeline

Tips

You can find SRR data so quickly in http://sra.dbcls.jp/

Q&A

• In exporting output.tsv to iDEP, which data type should I select?

When iDEP reads output.tsv, please put a check to Read counts data.

Issue

Please refer to issue

Releases

Please refer to Relases

• add support for udocker
• add setting of species
• gtf and transcript file from GENCODE
• salmon
• trimmomatic(legacy)
• trim_galore!
• tximport
• fastxtools(for Ion)
• judging fastq or SRR(manual)
• introduce "salmon gcbias correction"
• salomn validateMappings
• pigz(multithread version of gzip)
• fasterq-dump
• cwl development is in progress
• rename to "ikra"
• protein coding option

Legacy

Moved the flow using trimmomatic to ./legacy

Reference

• biocontainers : SNP-calling
• idep
• GENCODE
• salmon

Development of cwl ver.

2019/03/22 https://youtu.be/weJrq5QNt1M We tried developing it because Mr.Michael visited Japan. For now, cwlnized trim_galore and salmon in PE.

cd test/cwl_PE && bash test.sh

sorce and reference ー cwl_tools

• https://github.com/pitagora-galaxy/cwl
• https://github.com/roryk/salmon-cwl

Citation

Hiraoka Yu, Yamada Kohki, Ryuichiro Yamsasaki, YusukeKawasaki, Kitabatake Ryoko, Matsumoto Yasunari, Ishikawa Kaito, Umezu Yuto, Hirose Haruka, & Yoshiaki Yasumizu. (2021). yyoshiaki/ikra: ikra v2.0.1 (v2.0.1). Zenodo. https://doi.org/10.5281/zenodo.5541399

Licence (Updated in Ver. 2.0)

This software is freely available for academic users. Usage for commercial purposes is not allowed. Please refer to the LICENCE page. If you are not an academic user, please contact to the author.