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creating an MSA file for 20+ million reads whith non-directional sequences

Hi, I am trying to align about 20,000,000 reads that have been sequenced in a non-directional way. Each read is about 30bp, and I have a ref of 30bp. I tried using bowtie2, making an index of the ref, and ran it wil -L 5 (so the seed is smaller) and got 0% alignment What is the best way to do this? would you recomment Pagan for doing it? or is there another MSA program that can handle it (I know clustalW can't... can muscle or MAFFT maybe?) Thanks,

rna-seq rna msa
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written by blur blur

You can't do a MSA with a NGS aligner. If you have short sequences like that you should be using bowtie1 instead for alignment. Why do you want to create an MSA with 20M+ NGS reads?

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written by GenoMax GenoMax

genomax is right, but in addition I wonder why you are having a reference sequence of 30bp. I therefore think you are trying to do something odd.

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written by Michael Dondrup Michael Dondrup

These are synthetic oligos that are used as a test prior ro RNA-seq analysis, to check an hypothesis...

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written by blur blur

Then perhaps dedupe the dataset (dedupe.sh from BBMap should do it easily) before doing the MSA with that reduced dataset.

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written by GenoMax GenoMax

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