Question: Align to custom sequences using STAR aligner
0
gravatar for roy.granit
5 months ago by
roy.granit830
Israel/LabWorm
roy.granit830 wrote:

I have 3 amplicons that I have sequenced and now I wish to align them to the sequences of these three genes so I can quantify how many reads mapped to each gene.

In order to use STAR must I go through the steps of genome indexing or is there a way to align stright with the fasta file containing the 3 genes?

Thanks!

rna-seq star • 178 views
ADD COMMENTlink written 5 months ago by roy.granit830
1

you could make (and index) a custom file with only those 3 genes (perhaps go for the gene locus sequences, rather than CDS or such). Keep in mind their is a risk that you bias your analysis.

Personally I think STAR is fast enough to use the whole genome as reference and do some filtering after mapping.

ADD REPLYlink written 5 months ago by lieven.sterck8.9k

I did not mention that some of these genes do not exist in the genome..

ADD REPLYlink written 5 months ago by roy.granit830

If you don't expect off-target amplification then why not use whole genome indexes if they are available. That can also show you off-target amplification (if there was any).

If not, you will need to index the reference sequences (which should be trivial for a small number).

BBMap allows you to align directly without creating indexes first: bbmap.sh in=your.fq.gz ref=your_multifasta.fa out=aligned.bam

ADD REPLYlink modified 5 months ago • written 5 months ago by genomax92k

Thanks, how reliable is bbmap? never heard of it..

ADD REPLYlink written 5 months ago by roy.granit830
1

You are missing some important knowledge in that case :-) BBMap goes toe to toe with any NGS aligner out there. I will leave this link here so you can see what the BBTools suite can do (besides alignments). A guide for BBMap the aligner is available here.

ADD REPLYlink modified 5 months ago • written 5 months ago by genomax92k
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