I've just starting sequencing using MiSeq and am stumbling a bit in the analysis of my output reads, specifically using bowtie2 to align the reads to the reference genome. I'm using bowtie to get a rough estimate of what my amplification looks like and it's generally intuitive and quick. However:
When aligning reads to a genome sequence, what is “% reads align exactly 1 time” vs “% reads align >1 times” in the output? From my understanding, bowtie only records the best possible match (by default).
Is this saying that, to use an example from one of my samples, 40.26% of my reads align equally well to several places in the genome, suggesting a repeat region, while only 17.35% of my reads align to a unique region?
Or should this be interpreted as 40% of the reads align somewhere in the genome that already has one or more reads assembled to it, and that 17% of my reads are “unique”?
Also, my —-local alignments are substantially different (99% of reads align using —-local while only <70% without) from those where I do not specify —-local, even though I trim adapters and low quality reads before aligning (using trimmomatic). From my understanding of —-local, it should give a slightly more liberal alignment, but the two should be closer especially with trimming beforehand.
Forgive my ignorance and thanks for any advice.
Alex
When aligning reads to a genome sequence, what is “% reads align exactly 1 time” vs “% reads align >1 times” in the output? From my understanding, bowtie only records the best possible match (by default).
Is this saying that, to use an example from one of my samples, 40.26% of my reads align equally well to several places in the genome, suggesting a repeat region, while only 17.35% of my reads align to a unique region?
Or should this be interpreted as 40% of the reads align somewhere in the genome that already has one or more reads assembled to it, and that 17% of my reads are “unique”?
Also, my —-local alignments are substantially different (99% of reads align using —-local while only <70% without) from those where I do not specify —-local, even though I trim adapters and low quality reads before aligning (using trimmomatic). From my understanding of —-local, it should give a slightly more liberal alignment, but the two should be closer especially with trimming beforehand.
Forgive my ignorance and thanks for any advice.
Alex