pRNASeqTools

Integrated High-throughput Sequencing Data Analysis for Plant

Author: Dr. Chenjiang You

Current Version: 0.8

Latest updata: 02/09/2022

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Introduction

The pRNASeqTools is a Perl and R based pipeline designed for automatic general analysis for Illumina sequencing data in supported plants.

Currently it is able to process small RNA-seq, mRNA-seq, degradome-seq, CLIP-seq, ChIP-seq, and WGBS-seq and generally analyze them. See below for more information of specific tasks.

The phasiRNA identification module was contributed by Dr. Xuan Ma.

If you have any questions or comments, please submit an issue in the GitHub or directly email to Chenjiang You.

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Before the first run

To successfully run this pipeline on your own computer or server, several pieces of dependent software are needed. See INSTALL.md for detailed information.

For genome reference files, please contact Chenjiang You for pre-built genomes or the instructions for new genomes.

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Input files preparation

The only input files needed are Illumina output fastq files, either in the FASTQ format or corresponding compressed file formats .gz and .bz2. SRR accessions are also accepted. Note: the FASTA format is not supported. You may convert the fasta format to fastq format by adding artificial sequence names and qualities.

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Running pRNASeqTools

Usage and Options

See help information of pRNASeqTools simply by execute pRNASeqTools.

Quick examples

General analysis for small RNA-seq from samples control and treatment with 3 biological replicates

pRNASeqTools srna --adaptor AGATCGGAAGAGC --control control=control_1.fastq.gz+control_2.fastq.gz+control_3.fastq.gz --treatment treatment=treatment_1.fastq.bz2+treatment_2.fastq.bz2+treatment_3.fastq.bz2

Only mapping the small RNA reads to the genome and creating read count files

pRNASeqTools srna --adaptor AGATCGGAAGAGC --mapping-only --control control=control_1.fastq+control_2.fastq+SRRXXXXXXX

Perform statistic analyses in the folder containing pre-processed data

pRNASeqTools srna --nomapping --control control=3 --treatment treatment=3

General analysis for mRNA-seq from samples control and treatment with 3 biological replicates

pRNASeqTools mrna --control control=control_1.fastq.gz+control_2.fastq.gz+control_3.fastq.gz --treatment treatment=treatment_1.fastq.bz2+treatment_2.fastq.bz2+treatment_3.fastq.bz2

General analysis for paired mRNA-seq from samples control and treatment with 3 biological replicates

pRNASeqTools mrna --control control=control_1_R1.fastq.gz,control_1_R2.fastq.gz+control_2_R1.fastq.gz,control_2_R2.fastq.gz+control_3_R1.fastq.gz,control_3_R2.fastq.gz --treatment treatment=treatment_1.fastq.bz2+treatment_2.fastq.bz2+treatment_3.fastq.bz2

Trunction and tailing analysis of plant miRNAs

pRNASeqTools tt --adaptor AGATCGGAAGAGC --control control=control_1.fastq.gz+control_2.fastq.gz+control_3.fastq.gz --treatment treatment=treatment_1.fastq.bz2+treatment_2.fastq.bz2+treatment_3.fastq.bz2

Degradome data analysis

pRNASeqTools deg --adaptor AGATCGGAAGAGC --control control=control_1.fastq.gz+control_2.fastq.gz+control_3.fastq.gz --treatment treatment=treatment_1.fastq.bz2+treatment_2.fastq.bz2+treatment_3.fastq.bz2

Two-factor DE analysis

pRNASeqTools tf --control control=time1,3,time2,3 --treatment treatment=time1,3,time2,3

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Output

All output files are stored in the output directory. Mapping statistics are stored in the log file log_xxxxxxxxx.txt.

sRNA analysis

Several groups of files are generated in the output directory:

1. count files and nf files can be used for later --nomapping runs, which will not invoke the mapping procedures.

   The second to tenth columns of count files are numbers of assigned small RNAs with length 18 - 26nt.

2. pdf files showing the reproductivity of biological replicates and the relationship of samples.

3. csv files containing the results of statistic analyses, of which hyper and hypo files indicate the significant ones filtered out based on input parameters.

4. bedgraph files for visualization in IGV. Note: Keywords are embedded in the file names, indicating the targets and methods.

phased siRNA analysis

Trunction and tailing analysis

1. miRNA reads are categorized in the out files. The second column shows the number of tailed nucleotides and the third column shows the number of truncated nucleotides.
2. pdf files are bubble plots for each miRNA.

Degradome data analysis

1. bam files contain the mapped reads.
2. txt files report the identified peaks on each transcripts.

mRNA analysis

1. Mapped reads in bam files and read counts for each gene in txt files are reported.
2. Up-regulated and down-regulated DEG results are reported in total.hyper.csv and total.hypo.csv files.

Ribo-seq analysis

Two-factor of DEG analysis

1. This mode can only run in srna and mrna output folders.
2. Up-regulated and down-regulated DEG results are reported in total.hyper.csv and total.hypo.csv files.

CLIP-seq analysis

ChIP-seq analysis

WGBS-seq analysis

RiboMeth-seq analysis