CAB small RNA-seq Nextflow pipeline [V1.3.0] based on sRNAtools

CAB internal wikipage

https://wiki.stjude.org/display/CABI/CAB+miRNAseq+nextflow+pipeline

Dependencies

• databases: /research/groups/cab/projects/automapper/common/tchang1/Softwares/sRNAtools/db/ (defined by DBCONFIG.txt)
• bowtie-1.2.2 index: /research/groups/cab/projects/automapper/common/hjin/bin/bowtie-1.2.2/indexes
• modules: nextflow/21.10.5, R/4.3.2,perl/5.20.1, samtools/1.9 , bedtools/2.25.0, bowtie/1.2.2, trimgalore/0.6.6,fastqc/0.11.8, python37

Usage

# export LD_LIBRARY_PATH="/hpcf/authorized_apps/rhel8_apps/gcc/13.1.0/install/lib64:$LD_LIBRARY_PATH"
module nextflow/21.10.5
nextflow run sRNAtools.nf --help 


Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/research/rgs01/scratch_lsf/java -XX:ParallelGCThreads=1 -XX:ConcGCThreads=1
N E X T F L O W  ~  version 22.04.3
Launching `/research/groups/cab/projects/automapper/common/hjin/bin/cab_nextflow/sRNAtools/sRNAtools.nf` [naughty_blackwell] DSL2 - revision: cb50824d71

        Welocme to run Nextflow Pipeline sRNAtools.nf [version 1.3.0, 12/15/2024]
       Usage:
        A typical command for running the pipeline is as follows:
        nextflow run sRNAtools.nf -profile local --fqlist fq1.lst --Trim_Mode 1 --outdir run1 --prefix hendegrpq
        nextflow run sRNAtools.nf -profile local --fqlist fq2.lst --Trim_Mode 2 --outdir run2 --prefix sapkogrp

        Configurable arguments:
        --outdir                      The directory for the pipeline output
        --prefix                      output prefix for your analysis
        --A                           adapter sequence for read 1 if Trim_Mode is 3
        --fqlist                      Fastq file list that you want to call sRNAtools
                 format1 (2 columns) for single_end_oneLane:
                          SampleName L001_R1_001.fastq.gz
                 format2 (2 columns) for single_end_multiLanes:
                          SampleName L001_R1_001.fastq.gz,L002_R1_001.fastq.gz
        --species                    hsa or mmu (use hsa for human; mmu for mouse)
        --Trim_Mode                  Select a Small RNA-seq protocol
                1 NextFlex Small RNA-Seq Kit v3 (Hartwell Center protocl)
                2 NEBNext + sapkogrp/TrueSeq Index 26
                3 custom ; need to set adapter sequence by -A
        --sRNAtools_path        if you use your own sRNAtools installation
        --conda_env_path  if you create your own sRNAtools conda_env
        --help | h                    Show this usage statement.

       Note:
         All the above arguments can be configured by the command line interface or in the nextflow.config (default)

Input

A tab-delimited sample list text file is needed. There are two acceptable formats as follows.

• Format1 (2 columns) for single_end_oneLane.The first column is the SampleName and the second column is the fastq.gz filename of that sample.

Sample1	Sample1_L001_R1_001.fastq.gz
Sample2	Sample2_L001_R1_001.fastq.gz

• Format2 (2 columns) for single_end_multiLanes. If there are multiple lanes (fastq.gz files) per sample, concatenate multiple fastq.gz filenames by comma in the second column:

Sample1	Sample1_L001_R1_001.fastq.gz,Sample1_L002_R1_001.fastq.gz
Sample2	Sample2_L001_R1_001.fastq.gz,Sample2_L002_R1_001.fastq.gz,Sample2_L003_R1_001.fastq.gz

Output

• table.result.txt # raw counts table of all small RNA species (one file for each sample)
• all.count.txt /all.RPM.txt # merged raw counts /RPM table of all small RNA species (including all samples)
• miRNA.count.txt / miRNA.RPM.txt # merged raw counts /RPM table of miRNA (including all samples)

A toy example

input

cat fq1.lst 
R022_3P /research/groups/sapkogrp/projects/CAB/common/UMD_sRNA_seq/R022_3P_L001_R1_001.fastq.gz
R044_3P /research/groups/sapkogrp/projects/CAB/common/UMD_sRNA_seq/R044_3P_L002_R1_001.fastq.gz

run nextflow

cmd="nextflow run sRNAtools.nf -c nextflow_conda.config -profile cluster --fqlist fq1.lst --Trim_Mode 1 --species hsa --outdir sapkogrp --prefix sapkogrp"

output

└── sapkogrp
    ├── clean
    │   ├── R022-12P
    │   │   └── R022-12P_clean.fa
    │   └── R022-3P
    │       └── R022-3P_clean.fa
    ├── results
    │   ├── R022-12P_table.result.txt
    │   ├── R022-3P_table.result.txt
    │   ├── sapkogrp_hsa_all.count.txt
    │   ├── sapkogrp_hsa_all.RPM.txt
    │   ├── sapkogrp_hsa_miRNA.count.txt
    │   └── sapkogrp_hsa_miRNA.RPM.txt
    └── Trim_Galore
        ├── R022-12P
        │   ├── R022-12P_R1.fastq.gz_trimming_report.txt
        │   ├── R022-12P_trimmed_fastqc.html
        │   └── R022-12P_trimmed.fq
        └── R022-3P
            ├── R022-3P_R1.fastq.gz_trimming_report.txt
            ├── R022-3P_trimmed_fastqc.html
            └── R022-3P_trimmed.fq

sRNAtools

https://academic.oup.com/bib/article/22/1/463/5686255 http://rnainformatics.org.cn/sRNAtools/download.php

Maintainers

• Hongjian Jin

Update log

• 2023-04-18, Version 1.0.0. implement NEXTflex small RNAseq protocol
• 2023-04-20, Version 1.2.0. implement NEBNext small RNAseq protocol
• 2024-11-16, Version 1.3.0. check compatibility with rhel8; minor changes
• 2024-12-15, finished internal test.