Mapping (with HISAT2 / Bowtie) on custom genomes yields always 0 matches

Question: Mapping (with HISAT2 / Bowtie) on custom genomes yields always 0 matches

3 months ago by

Hi,

I've the following problem. I'm using HISAT2 and Bowtie. I'm using a gene with 1200 nucleotides which is present in the hg38. When aligning my FASTQ data that ist around 250 nucleotides to the hg38 I get 99%+ mapping on the that gene, when I use the gene itself as a FASTA genome for mapping I get 0% mapping. The reads are all in-between those 1200 bases and if I run a blast on some of the reads I've a 100% match. Can someone explain to me what I'm doing wrong?

I can see that the script recognized the gene that I'm using as a genome:

@SQ SN:HPD_xdna_1182    LN:1182

There seems to be the issue?! Even if I rename it.

Dataset peek HISAT2 summary stats:

Total reads: 86715

    Aligned 0 time: 86715 (100.00%)

    Aligned 1 time: 0 (0.00%)

    Aligned >1 times: 0 (0.00%)

alignment dna rna galaxy spliced • 180 views

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modified 3 months ago • written 3 months ago by stoyan.velkov • 10

3 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

It sounds like you need to tune the alignment parameters. Spliced mapping settings, overlap, overhang, mismatches, and the like. I suspect the current settings are too strict.

And just in case, this is the FAQ for how to format a custom reference genome (same rules apply to transcriptome data): https://galaxyproject.org/support/ >> Preparing and using a Custom Reference Genome or Build

Spliced mapping:

Query RNA, target DNA (genome or full transcript/gene bound including introns)

Unspliced mapping:

Query RNA, target RNA (transcript only mRNA, no introns)
Query DNA, target RNA
Query DNA, target DNA

Exception: most bacterial genomes would be unspliced for all RNA/DNA combinations.

Thanks! Jen, Galaxy team

ADD COMMENT • link modified 3 months ago • written 3 months ago by Jennifer Hillman Jackson ♦ 25k

3 months ago by

stoyan.velkov • 10

stoyan.velkov • 10 wrote:

Hi Jen,

Thanks for your answer. So far I only had done that:

Did you use NormalizeFasta with the options to wrap sequence lines at 80 bases and to trim the title line at the first whitespace?

Will continue to try, I tried also with different settings, but wasn't lucky so far. Atleast I know now what could be the possible issues to look for. I'm looking for DNA query on DNA

Greetings, Stoyan

ADD COMMENT • link written 3 months ago by stoyan.velkov • 10

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