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I am wondering if anyone has tried aligning 1d^2 RNA-seq data. I am trying that on minimap2. It is giving me very low ~50% of mapped reads. it gives ~95% mapped reads for 1D.

I am using the following command for 1d and 1d^2:

minimap2 -ax splice -uf -k14 ref.fa direct-rna.fq > aln.sam

Update:

I also tried to play around the command using :

minimap2 -ax splice -k14 -a ref.fa direct-rna.fq > aln.sam
minimap2 -ax splice -ur -k14 ref.fa direct-rna.fq > aln.sam
minimap2 -ax splice -ub -k14 ref.fa direct-rna.fq > aln.sam

It did not work better.

FYI: I am trying to find answer for this from few days. I have posted this question on Biostars before. I did not get any response. Hence, I am trying to find the answer here.

Thank you in advance. I really appreciate any help.

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    $\begingroup$ Have you tried blatting/blasting a few of the non-aligning reads to check if they're okay? $\endgroup$
    – Wouter De Coster
    Mar 2, 2018 at 8:52
  • $\begingroup$ No, I have not. But it will be good exercise. I will do it. Thanks. $\endgroup$
    – KVC_bioinfo
    Mar 2, 2018 at 15:18
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    $\begingroup$ You might consider trying to put the blast results into MEGAN to see if there is contamination in your sample. Also, could you please update us with what your sample type is (organism, tissue, origin), and if you have an amplicon, gDNA, cDNA library? Here is the link to MEGAN: ab.inf.uni-tuebingen.de/software/megan6 $\endgroup$
    – conchoecia
    Mar 5, 2018 at 17:46
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    $\begingroup$ I have a similar problem (many unaligned reads). Did you find a solutioin? By any chance, did you compare minimap2 results with graphmap? $\endgroup$
    – mjoppich
    Jun 30, 2018 at 17:06
  • $\begingroup$ Please consider the answer @KVC_bioinfo and if you're happy with it for upvoting/accepting. Its good for site stats. $\endgroup$
    – M__ ♦
    Sep 15 at 20:48

1 Answer 1

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Since this is an old unanswered question where a lot of method development has since happened, going to put some answers here:

  • This rather old tool also mentioned in comments (graphmap) is supposedly good for this, though it may not be relevant anymore.
  • This paper uses minimap (not minimap2) alignments as a heuristic for read quality, suggesting that minimap2 should be good enough.

Note that depending on how you have processed (specifically basecalled) the data, 1d2 "second" reads may have low accuracy. That matches your ~50% unaligned proportion. You may need to do additional work during basecalling to fully use 1d2, as discussed in this paper.

Note: I am not an expert on nanopore and I put this answer together using google. Others are welcome to edit/editorialize.

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