The STAR error looks like this:
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EXITING because of FATAL ERROR in reads input: quality string length is not equal to sequence length
@SRR22XXXX.85715
TTAATGGTTGTCGTATGATATTTGTTACGATAATGAGGCTTTTTGTGATAGAAATATCATTAATGTTAATAATTGTAGGTGTAGAGATAAAGGAGG...
SOLUTION: fix your fastq file
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Inspired by my colleague’s work, I tried hisat2:
First download the source code, decompress and make.
Similar to other aligners, you need to build the genome index first:
hisat2-build -f genome.fasta index_name
It takes about 26 min for a 360M genome.
Then the run command is like this:
bin/software/hisat2-2.1.0/hisat2 -p 8 hisat2-index/index_name -U reads2map.fastq -q -S outfile.sam
Where as explained in the manual p is for threads, -U indicates files containing unpaired reads to be aligned, -q for fastq input, and -S for sam output.
The mapping is also pretty fast.
For counting the mapped reads, it’s common to use htseq-count, but it is slow. I tried another tool “featureCounts”, which also uses a gtf annotation:
featureCounts -t exon -g gene_id -a annotation.gtf -o counts_out.txt outfile.sam
You can also use “-s 2” for reversely stranded.
The counting is super fast, within < 1min you can get your count file for downstream analyses. So there is no need to submit a job to a cluster. Below is the hilarious output interface:
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========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
v1.4.5-p1
//========================== featureCounts setting ====================\\
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|| Input files : 1 SAM file ||
|| S SRR2245469egg-v7.sam ||
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|| Output file : Eggcounts-featureCounts_s1.txt ||
|| Annotations : V7_r7.gtf ||
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|| Threads : 1 ||
|| Level : meta-feature level ||
|| Paired-end : no ||
|| Strand specific : yes ||
|| Multimapping reads : not counted ||
|| Multi-overlapping reads : not counted ||
|| ||
\\===================== https://subread.sourceforge.net/ ===============//
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