Hi everyone,
I'm preparing a ddRAD-seq plate for Illumina HiSeq 4000 sequencing and I'd like to know if anyone has seen similar Bioanalyzer traces post-size selection with the BluePippin. The first image below shows my traces post SS (left) and pre-SS (right). In our lab, we clean up the DNA before PCR but after digestion/ligation, and size selection is performed post-PCR.
I used a fairly broad size selection window of 300-500 bp. The Bioanalyzer trace of my library before size selection shows a fairly even fragment size distribution, though with a slight bias towards larger fragment sizes. However, after size selection, the Bioanalyzer trace shows a sharp peak rather than a plateau between 300-500 bp. For comparison, I also included a photo of a Bioanalyzer trace of a different post size-selection ddRAD library a lab mate produced several months ago.
Has anyone experienced a similar issue before? I'm assuming sequencing will produce sufficient reads from this plate, but I am anticipating more plates of these species in the future and I'd like to combine datasets with similar read distributions (i.e. ideally I would have a plateau distribution from 300-500 bp post-size selection).
Thank you!
I'm preparing a ddRAD-seq plate for Illumina HiSeq 4000 sequencing and I'd like to know if anyone has seen similar Bioanalyzer traces post-size selection with the BluePippin. The first image below shows my traces post SS (left) and pre-SS (right). In our lab, we clean up the DNA before PCR but after digestion/ligation, and size selection is performed post-PCR.
I used a fairly broad size selection window of 300-500 bp. The Bioanalyzer trace of my library before size selection shows a fairly even fragment size distribution, though with a slight bias towards larger fragment sizes. However, after size selection, the Bioanalyzer trace shows a sharp peak rather than a plateau between 300-500 bp. For comparison, I also included a photo of a Bioanalyzer trace of a different post size-selection ddRAD library a lab mate produced several months ago.
Has anyone experienced a similar issue before? I'm assuming sequencing will produce sufficient reads from this plate, but I am anticipating more plates of these species in the future and I'd like to combine datasets with similar read distributions (i.e. ideally I would have a plateau distribution from 300-500 bp post-size selection).
Thank you!
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