ikra v1.2.1 -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.
日本語ドキュメントはこちら
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.
-t, --threads
-o, --output output file. (default : output.tsv)
-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.
- 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.
experiment matrix should be separated by commas (csv format).
SRR mode
name | SRR | Layout | condition1 | ... |
---|---|---|---|---|
Treg_LN_1 | SRR5385247 | SE | Treg | ... |
Treg_LN_2 | SRR5385248 | SE | Treg | ... |
fastq mode
name | fastq(PREFIX) | Layout | condition1 | ... |
---|---|---|---|---|
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
andfastq.gz
. - fastq file specifies path excluding
fastq.gz
or_1.fastq.gz
and_2.fastq.gz
. For example,hoge/SRR5385247.fastq.gz
is described ashoge/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.
Bug 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.
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
test
Illumina trim_galore ver.
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
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/
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
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
Citation
Hiraoka, Y., Yamada, K., Kawasaki, Y., Hirose, H., Matsumoto, K., Ishikawa, K., & Yasumizu, Y. (2019). ikra : RNAseq pipeline centered on Salmon. https://doi.org/10.5281/ZENODO.3352573