Question: Alignment of targeted sequencing to the whole genome or to targeted reference
0
gravatar for biobiu
13 months ago by
biobiu110
United States
biobiu110 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 • 645 views
ADD COMMENTlink modified 12 months ago • written 13 months ago by biobiu110

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 12 months ago by biobiu110
2
gravatar for finswimmer
13 months ago by
finswimmer13k
Germany
finswimmer13k 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 13 months ago by finswimmer13k

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 13 months ago by Charles Warden7.5k
0
gravatar for Bo Wang
13 months 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 13 months 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 13 months ago by ATpoint28k
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