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Question: Using unaligned reads as input for trinity
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'I have done HiSAT mapping for my RNAseq sample and I have got unaligned FASTQ sanger file. Can i use this unaligned files as input for running trinity or Should i use the original SRA reads file.

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What you should do is to take some of those unaligned reads and check them using blast. At a minimum there may be contamination in data you are using. If so there would be no point in wasting further time on this dataset.

If the reads are from the right genome then you should spend some time figuring out why HISAT2 is unable to align them.

ADD REPLYlink 10 months ago
genomax
68k
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Thank you. I'll check that.

ADD REPLYlink 10 months ago
Pranavathiyani G
• 230
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In addition to what genomax proposes, I would also just support your approach, or better simply running a full Trinity assembly. It doesn't cost you much except some CPU time and after that you can simply blast all the assembled transcripts. More generally speaking, whenever problems in an analysis arise, one should try out a bunch of different things and make some hypothesis about what is going wrong. Btw, what is your definition of a low mapping rate?

  • wrong genome
  • draft genome with lots of gaps, unmapped regions
  • contamination
  • ribosomal RNA
  • failed sequencing or protocol
ADD REPLYlink 10 months ago
Michael Dondrup
46k
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Well technically you can anything you wish. Feed the software with something and it will spit out something more but why do want to do that ?

Share your objective here.

ADD REPLYlink 10 months ago
Vijay Lakhujani
4.1k
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Because the result of HISAT mapping was low.

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Pranavathiyani G
• 230
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You should always mention that in your post along with mapping statistics. Anyways, look at this old post, there is a long discussion on similar problem I had faced couple of months back!

Too low mapping percentage using HISAT2 on human reference genome.

ADD REPLYlink 10 months ago
Vijay Lakhujani
4.1k
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Thanks. I'll have a look at it.

ADD REPLYlink 10 months ago
Pranavathiyani G
• 230

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