Cross-Posting from BioStars. Not sure if theres a better sub-forum, sorry if out of place!
have been trying to read and find out more about the pitfalls of the topic here but have not been able to find many good, straightforward answers or validations on the problems that would happen if one did try to do exome capture and sequencing when using mRNA sequences to design probes instead of exon sequences. I have seen papers refer to specifically trying to avoid introns when doing this type of reduced representation sequencing, but not real explanation as to why.
For context, I have inherited data that used Agilent designed probes for exome capture and sequencing. However, when I was examining the probe sequences, I noticed that when mapped to the reference genome, some of them covered regions larger than expected. It turned out that was because there are introns within the genome at those locations. The probes, however, do not account for the intron. Example: If the genomic region was ACTGAGACT[GAATAAG]CTACGATA, with the intron in brackets... the probe for this region is ACTGAGACTCTACGATA
When aligning the sequencing data that was generated from sequencing the DNA that was captured on the probes, there are indeed reads that map to the genomic sequence properly, including the intronic sequences. This suggests that despite not being within the probe design, the introns were still captured.
My main concerns are as follows:
I get the sense that this decision might have unintended consequences, and I have a partial shape in my head of why this might be problematic during the capture phase. But what is done is done, so I am trying to understand, with the data that was generated from this, if there are problems I need to be aware of.
Thanks for any help or insights people may have!
have been trying to read and find out more about the pitfalls of the topic here but have not been able to find many good, straightforward answers or validations on the problems that would happen if one did try to do exome capture and sequencing when using mRNA sequences to design probes instead of exon sequences. I have seen papers refer to specifically trying to avoid introns when doing this type of reduced representation sequencing, but not real explanation as to why.
For context, I have inherited data that used Agilent designed probes for exome capture and sequencing. However, when I was examining the probe sequences, I noticed that when mapped to the reference genome, some of them covered regions larger than expected. It turned out that was because there are introns within the genome at those locations. The probes, however, do not account for the intron. Example: If the genomic region was ACTGAGACT[GAATAAG]CTACGATA, with the intron in brackets... the probe for this region is ACTGAGACTCTACGATA
When aligning the sequencing data that was generated from sequencing the DNA that was captured on the probes, there are indeed reads that map to the genomic sequence properly, including the intronic sequences. This suggests that despite not being within the probe design, the introns were still captured.
My main concerns are as follows:
- Will this affect the accuracy of the reads that were captured and the sequencing? Could this lead to something akin to chimeras? Or, is the downside of having the introns not accounted for a question of coverage and yield over coding regions?
- Would this affect SNP calling in any capacity? Part of the purpose of this data set was for calling SNPs on the data, and I am worried that this aspect of the probe design being fraught is going to affect that.
- Is this a non-issue for introns under/over a certain size?
I get the sense that this decision might have unintended consequences, and I have a partial shape in my head of why this might be problematic during the capture phase. But what is done is done, so I am trying to understand, with the data that was generated from this, if there are problems I need to be aware of.
Thanks for any help or insights people may have!
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