Question: Alignment of targeted sequencing to the whole genome or to targeted reference
0
gravatar for biobiu
7 weeks ago by
biobiu10
United States
biobiu10 wrote:

I've sequenced targeted DNA panel. I'm aligning it using bwa mem once to the whole genome and twice to the targeted sequences. When aligning it to the reference genome I'm getting ~30% of the reads mapped to the targeted regions. However, when aligning it to the panel sequences 70% of the read are aligned successfully.

Any idea how can it happen? even if there is favored alignment in the whole genome these reads should be assigned as supplementary alignment? Isn't it?

alignment • 170 views
ADD COMMENTlink modified 4 weeks ago • written 7 weeks ago by biobiu10

Thanks for all responses.

I understand why aligning to the whole genome is recommended.

However, I accepted that most of the reads will still align to the targets when aligning to the whole genome but will be assigned as secondary. It seems that this is not the case here....

ADD REPLYlink written 4 weeks ago by biobiu10
2
gravatar for finswimmer
7 weeks ago by
finswimmer9.9k
Germany
finswimmer9.9k wrote:

Hello,

there are only secondary alignments (not supplementary) if the probability of these alignments are similar. So in your whole genome alignment lots of your reads maps significantly better to other regions, than the one you guess.

That's a very good example why you should always align to the whole genome, even if you use panels.

fin swimmer

ADD COMMENTlink written 7 weeks ago by finswimmer9.9k

Yes - I agree.

In fact, further filtering the variants and/or alignment to just the target regions (after initially allowing reads to be aligned to other regions) should help with reducing false positives.

ADD REPLYlink written 7 weeks ago by Charles Warden6.1k
0
gravatar for Bo Wang
7 weeks ago by
Bo Wang0
China/Shang hai/WuXi AppTec
Bo Wang0 wrote:

The sequences of the target regions of your panel is not unique. So always align to the whole genome, or you have to confirm that the primers of the panel are unique.

ADD COMMENTlink written 7 weeks ago by Bo Wang0

Even if the capturing oligos were unique/specific in silico (which they typically are otherwise the kit or design would be of poor quality), this does not ensure perfect capturing efficiency and absence of off-target captures during the experiment. Therefore, as you say, align against the entire reference genome.

ADD REPLYlink written 7 weeks ago by ATpoint13k
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