error in rmats when mapping

Question

[katie326]

I'm trying to mRNA alternative splicing analysis with rmats and am encountering an error

My input code was:

rmats.py --s1 /Users/deaw/Desktop/Coding/s1.txt --s2 /Users/deaw/Desktop/Coding/s2.txt --gtf /Users/deaw/Desktop/Coding/Homo_sapiens.GRCh38.105.gtf --bi /Users/deaw/Desktop/Coding/STAR_binary_index -t paired --readLength 100 --nthread 4 --od /Users/deaw/Desktop/Coding/output --tmp /Users/deaw/Desktop/Coding/tmp_output

This was the output:

mapping the first sample mapping sample_0, /Users/deaw/Desktop/Coding/1_1.R1.fastq /Users/deaw/Desktop/Coding/1_1.R2.fastq is done with status -9 error in mapping sample_0, /Users/deaw/Desktop/Coding/1_1.R1.fastq /Users/deaw/Desktop/Coding/1_1.R2.fastq: -9 error detail: STAR --chimSegmentMin 2 --outFilterMismatchNmax 3 --alignEndsType EndToEnd --runThreadN 4 --outSAMstrandField intronMotif --outSAMtype BAM SortedByCoordinate --alignSJDBoverhangMin 6 --alignIntronMax 299999 --genomeDir /Users/deaw/Desktop/Coding/STAR_binary_index --sjdbGTFfile /Users/deaw/Desktop/Coding/Homo_sapiens.GRCh38.105.gtf --outFileNamePrefix /Users/deaw/Desktop/Coding/tmp_output/2022-01-11-17_05_09_640501_bam1_1/ --readFilesIn /Users/deaw/Desktop/Coding/1_1.R1.fastq /Users/deaw/Desktop/Coding/1_1.R2.fastq STAR version: 2.7.9a compiled: :/Users/cshl/data/STAR/STAR/source Jan 11 17:05:09 ..... started STAR run Jan 11 17:05:09 ..... loading genome Jan 11 17:06:02 ..... processing annotations GTF Jan 11 17:06:36 ..... inserting junctions into the genome indices Jan 11 17:07:48 ..... started mapping Traceback (most recent call last): File "/Users/deaw/opt/anaconda3/envs/myenv/bin/rmats.py", line 595, in <module> main() File "/Users/deaw/opt/anaconda3/envs/myenv/bin/rmats.py", line 558, in main args = get_args() File "/Users/deaw/opt/anaconda3/envs/myenv/bin/rmats.py", line 203, in get_args args.b1, args.b2 = doSTARMapping(args) File "/Users/deaw/opt/anaconda3/envs/myenv/bin/rmats.py", line 81, in doSTARMapping raise Exception() Exception

Any help would be much appreciated Thanks

Katie

Answer 1

[katie326]

Some more info in case it helps:

The RNA-seq files I am analysing are fastq raw sequencing data taken directly from the encode project for shRNA knockdown followed by RNA-seq (shRNA RNA-seq) with control samples and experiment samples.

The gtf file I used was from the ensembl and was this file: Homo_sapiens.GRCh38.105.gtf.gz

The binary star index I assembled from this Homo_sapiens.GRCh38.105.gtf.gz from ensembl and Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz from ensemble

This was done with --genomeSAsparseD 3 --genomeSAindexNbases 12 as I don't have enough ram would this perhaps cause an issue in rmats?