RNA-Seq-annotation-and-comparison

Olson_lab repository

Augustus_gene_predict_RNA_seq_data repository: augustus_beocat.txt -

autoAugPred.pl -

intron_filter.pl -

shellForAug -

KSU_bioinfo_lab

Cite as: Jennifer Shelton et al.. (2015). RNA-Seq-annotation-and-comparison: RNA-Seq-annotation-and-comparison Version 1.0.0. Zenodo. 10.5281/zenodo.17589

###Count_fastas.pl

Count_fastas.pl - see assembly_quality_stats_for_multiple_assemblies.pl

###RNA-SeqAlign.pl

SYNOPSIS

RNA-SeqAlign.pl/RNA-SeqAlign.pl - The script writes scripts and qsubs to generate count summaries for illumina paired end reads after mapping against a de novo transcriptome. The script 1) converts illumina headers if the "-c" parameter is used, 2) cleans raw reads using Prinseq http://prinseq.sourceforge.net/manual.html, 3) creates a filtered transcriptome fasta file with putative transcripts less than 200 bp long removed and then indexes this transcriptome for mapping, 4) reads are then mapped to the length filtered de novo transcriptome using Bowtie2 in the best mapping default mode, read more about Bowtie2 at http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml, 5) count summaries are generated as a tab separated list where the first row is the sample ids and the first column is the name of the contig and the other values are the read counts per sample, see https://github.com/i5K-KINBRE-script-share/RNA-Seq-annotation-and-comparison/tree/master/KSU_bioinfo_lab/Count_reads_denovo for details on how reads are summarized.

For examples and parameter details run "perl RNA-Seq_align.pl -man" or visit https://github.com/i5K-KINBRE-script-share/RNA-Seq-annotation-and-comparison/blob/master/KSU_bioinfo_lab/RNA-SeqAlign/RNA-SeqAlignLAB.md. For other NGS Beocat Pipelines visit http://i5k-kinbre-script-share.github.io/transcriptome-and-genome-assembly/.

###RNA-SeqAlign2Ref.pl

RNA-SeqAlign2Ref.pl - The script writes scripts and qsubs to generate count summaries for illumina paired end reads after mapping against a reference genome. The script 1) converts illumina headers if the "-c" parameter is used, 2) cleans raw reads using Prinseq http://prinseq.sourceforge.net/manual.html, 3) index the reference genome for mapping, 4) reads are aligned to the genome with Tophat2 (read more about Tophat2 at http://tophat.cbcb.umd.edu/manual.html) and expressed genes and transcripts are assembled with Cufflinks2, 5) these assemblies are merged with Cuffmerge and differential expression is estimated with Cuffdiff2.

For examples and parameter details run "perl RNA-SeqAlign2Ref.pl -man" or visit https://github.com/i5K-KINBRE-script-share/RNA-Seq-annotation-and-comparison/blob/master/KSU_bioinfo_lab/RNA-SeqAlign2RefREADME.md. For other NGS Beocat Pipelines visit http://i5k-kinbre-script-share.github.io/transcriptome-and-genome-assembly/.

assembly_quality_stats_for_multiple_assemblies.pl - This script runs a slightly modified version of Joseph Fass' Count_fasta.pl (original available at http://wiki.bioinformatics.ucdavis.edu/index.php/Count_fasta.pl ) on a fasta file from each assembly. It then creates comma separated file called assembly_metrics.csv listing the N25,N50,N75, cumulative contig length, and number of contigs for each assembly (also download Count_fastas.pl and change $path_to_Count_fastas on line 13 of assembly_quality_stats_for_multiple_assemblies.pl).

USAGE: perl assembly_quality_stats_for_multiple_assemblies.pl [FASTA filename or filenames]

###Blastx.pl

Blastx.pl - Script outputs fasta records split into files of 100 or less sequences in a directory called split. It also creates blastx bash scripts and qsub commands. FindFailed.pl can be run after Blastx.pl to find fasta sequences that have not been blasted (e.g. when a running blastx times out).

For examples and parameter details run "perl Blastx.pl -man" or visit https://github.com/i5K-KINBRE-script-share/RNA-Seq-annotation-and-comparison/blob/master/KSU_bioinfo_lab/Blastx/Blastx_LAB.md. For other NGS Beocat Pipelines visit http://i5k-kinbre-script-share.github.io/transcriptome-and-genome-assembly/.

###tBlastx.pl

tBlastx.pl - Script takes a list of FASTA files and outputs bash scripts and qsub commands that run tBlastx on them against the NCBI "nt" database. These scripts will output tab delimited Blast results that include taxanomic information.

###Count_reads_denovo.pl

Count_reads_denovo.pl - This script takes sam files from Bowtie2 (one per biological or technical replicate) and outputs tab separated list where the first column is the name of the contig and the values are the read counts per sample (in the same order as you listed your sam files).

To use this script:

Step 1: Align paired end reads to a clustered de novo transcriptome using the Bowtie2 "best mapping" (default) reporting mode.

Step 2: Run by passing your SAM file output as arguements when you run Count_reads_denovo.pl (one SAM file per sample) in the same order as you enter your sample ids (ids are optional). A more detailed README can be viewed at https://github.com/i5K-KINBRE-script-share/RNA-Seq-annotation-and-comparison/tree/master/KSU_bioinfo_lab/Count_reads_denovo. The manual can be viewed by running:

perl Count_reads_denovo.pl --man

Reads are filtered based on MAPQ and pair relationships from Bowtie2 sam files. Reads passing these filters are counted as indicated below: Reads diagramed below would not pass the filters and would not be counted for any contig: Below is the decision diagram for the read counting script. Concordant pairs (CP), discordant pairs (DP), unpaired mates (UP), and mateless reads (UU) are defined in Bowtie2 documentation http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#sam-output :

Minimum MAPQ can be adjusted. The default minimum is 10.