I have pair-ended illumina samples with the read length of 100-150bp. I tried to map them to the reference genome/transcritpome, but almost nothing mapped using BWA or Bowtie.
When I blast them to nt reference using megablast, the most reads map to the right species. What does this mean? How can I interpret this? and what do I do to improve short-read alignment using BWA or bowtie?
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BLAST is a local aligner, bwa/bowtie attempt a semi-global alignment. Evaluate your BLAST alignments and look for patterns.
For example if all of your reads contain an adapter at the beginning or end, or if your reads contains lots of errors towards their end then bwa/bowtie will be unable to map them in a semi-global fashion, whereas BLAST can still align them in a local way.
Hey, just from your answer, is there any specific reason of adapter sequences being present in the middle of the reads or they are always at start or end.
it is about library preparation, if someone needs to sequence short miRNAs for example then they could go about this by ligating a known adapter that will be observed after the 20 or so bases, then they could add random bases after this adapter - the net effect thus is having an adapter in the middle and random bases after that
Yeah got ur point, but for some reason, I observe the 6 bp Illumina Tru-Seq adapters in the middle of ChIP-Seq reads, which I am not able to figure out why. Thanks Istvan
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Your question is very vague and people will probably be unable to help without further information.
What species? What type of experiment? How exactly did you run BWA and Bowtie? Can you give us some examples of reads that mapped with megablast and failed with BWA or Bowtie? What have you tried so far?