Question: problem with the alignment of RNAseq data
0
gravatar for Sara
2.7 years ago by
Sara90
Sara90 wrote:

I have some RNAseq data and trying to align them to the genome (hg19) using both STAR and BWA separately. but the problem is that for each file it should take at least half an hour but the strange thing is that for these files it takes at most 6 minutes and at the end when I visualize the bam files on UCSC or IGV for many genes I do not have enough reads (in fact very few reads which is not really normal based on my experience). do you know what the possible problem is?

rna-seq • 828 views
ADD COMMENTlink modified 2.5 years ago by Biostar ♦♦ 20 • written 2.7 years ago by Sara90
2

Hi Sara,

Please don't forget to follow up on your other thread: how to visualize the RNAseq data on IGV

Cheers, Wouter

ADD REPLYlink written 2.7 years ago by WouterDeCoster43k

Can you give details on number of reads, read length distribution, read quality distribution, the type of pairing (single- or paired-end sequencing), and anything else you've got? These can be quickly generated by using Fast QC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

It's neither a major issue, but BWA is not what I would initially think of for RNA-seq data. There are modern 'pseudo-aligners' like Kallisto, which can align large samples in just a few minutes each, but they align to a reference transcriptome in FASTA.

ADD REPLYlink written 2.7 years ago by Kevin Blighe60k

It's neither a major issue, but BWA is not what I would initially think of for RNA-seq data.

Except perhaps if OPs organism (important information left out) is prokaryotic?

ADD REPLYlink written 2.7 years ago by WouterDeCoster43k

It's hg19. Mentioned in the question. In addition try to count the number of reads mapped to the genome.

ADD REPLYlink written 2.7 years ago by Asaf7.6k

Ow yeah, missed that. Then bwa is not appropriate.

ADD REPLYlink written 2.7 years ago by WouterDeCoster43k

There are many "possible problems". Better if you troubleshoot the alignment. For starters, what is your mapping rate? STAR has some nice and informative log with this information and more. How many reads on the fastqs?

ADD REPLYlink written 2.5 years ago by h.mon29k
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