Hi all,
I have come across very peculiar alignment results for plant RNASeq data. I have aligned RNASeq data 150 *2 illumina stranded library [think matters only during expression quantification]. Aligned to its reference genome (https://www.ncbi.nlm.nih.gov/assembly/GCA_000826755.1) using bowtie2, tophat2, hisat2 and STAR (all run with default parameters. Same parameters of bowtie2 were used in tophat and hisat). It was interesting to see bowtie out performs splice aware aligner (expect STAR which wins marginally). Here are the stats (%),
Bowtie2- 53.57
tophat2 - 12.5
hisat2 - 39.24
STAR - 58.64
Similar post were there 1, 2 and could not find a conclusive result. From unaligned reads, 5 random set of 100 reads were checked, 60% of the sequences found match in blast result of which 72.09 are still matching to predicted mRNAs of the reference genome.
My questions, 1) Star being local aligner, how come these reads are missed out? (is it because long exon-exon junction gap)
2) How bowtie is performing better than splice aware programs?
Better? I'll write an aligner which will align 100% of the reads for you, but with a very low accuracy.
Nope.
I was just curious to know how bowtie performed better than splice aware aligners. header i meant in fun way
Sure, but the percentage aligned doesn't make it better, since it doesn't say anything about how correct the reads are. Perhaps Bowtie aligns to one exon only, and splice information is completely lost?
As per Wouter, the % alignment is not the complete story. You have neither given us data for BWA (even though I would use neither Bowtie2 nor BWA on their own for RNA-seq reads).
True. I wanted to have quick view on the data so choose bowtie, when % was only 53 then i went for splice aligners (tophat and hisat) those results were much lesser (was expecting it to be better than bowtie). When i checked unaligned, they are still matching to jujube. is it the data which is a questionable?? Here unaligned reads from star.
Thank you WouterDeCoster and Kevin Blighe for your valuable suggestions