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RobertsLab / paper-Olurida-gene

Gene expression data and R scripts for analysis
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Presence of SNPs in priming sites #9

Closed kubu4 closed 7 years ago

kubu4 commented 7 years ago

Please comment on whether the priming sites for the primers were known to contain SNPs that may have affected hybridization efficiency. This should be available since the Authors’ lab appears to have made its own transcriptome and have access to raw sequencing reads.

sr320 commented 7 years ago

FFS

sr320 commented 7 years ago

Priming sites are not known to have SNPs. The transcriptome was generated from a different population of oysters thus would not be directly informative. We cannot completely exclude the possibility of SNPs in the oysters examined. This could be determined with targeted high-throughput sequencing or targeted Sanger sequencing, both beyond the scope of this work.

kubu4 commented 7 years ago

I need to read a bit more, but is it possible this was actually checked this out via Sanger sequencing already??

http://heareresearch.blogspot.com/2015/06/6-23-2015-flanking-primer-pcr-pt-3.html

http://onsnetwork.org/kubu4/2015/06/25/sample-submission-olympia-oyster-pcrs-sanger-sequencing-2/

kubu4 commented 7 years ago

I've added the following section to address impact of SNPs in primer binding sites, but I'm not sure if this requires supporting data or not:

Primer binding sites were assessed for the presence of single nucleotide polymorphisms (SNPs) via Sanger sequencing. The majority of primer binding sites did not contain any SNPs. Those that did had only a single SNP and did not appear to impact qPCR data, as there was no noticeable difference in qPCR efficiencies in individuals having a SNP within a primer binding site for a given target.

kubu4 commented 7 years ago

Change to say something about Sanger sequencing of subset of primer binding sites.