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Hi all,
I'm making barcoded human exome libraries using Agilent's 50Mb all exon liquid phase SureSelect kit (following the SOLiD multiplexing protocol v1.3).
I have used Nimblegen for exon enrichment in the past and have found their qPCR a good way to estimate fold-enrichment before sequencing. Has anyone ever used the same primers to QC their Agilent libraries? What kind of results did you get for your Agilent libraries compared to Nimblegen? I used it to QC my first two libraries (following the v1.3 protocol) and the efficiency was quite poor.
I've also found different recommendations for the insert size. The v1.3 protocol is written for a 150-160bp insert size selection (assuming the truncated adapters are ~40-50bp total pre-capture), whereas a "tips and tricks" document from Agilent for non-barcoded libraries says to select for a 180bp insert size. On the other hand the Illumina protocol seems to produce libraries sized 250-500bp (no gel-based size selection). I was wondering what insert sizes people used for their capture, and if anyone has discovered which sizes capture most efficiently? Agilent seem to be changing their minds about what the optimal sizing is, which is concerning... I'd rather not go through a trial and error process for this as it is quite expensive and time consuming...
Any help or suggestions you can give would be hugely appreciated!
Thanks.
I'm making barcoded human exome libraries using Agilent's 50Mb all exon liquid phase SureSelect kit (following the SOLiD multiplexing protocol v1.3).
I have used Nimblegen for exon enrichment in the past and have found their qPCR a good way to estimate fold-enrichment before sequencing. Has anyone ever used the same primers to QC their Agilent libraries? What kind of results did you get for your Agilent libraries compared to Nimblegen? I used it to QC my first two libraries (following the v1.3 protocol) and the efficiency was quite poor.
I've also found different recommendations for the insert size. The v1.3 protocol is written for a 150-160bp insert size selection (assuming the truncated adapters are ~40-50bp total pre-capture), whereas a "tips and tricks" document from Agilent for non-barcoded libraries says to select for a 180bp insert size. On the other hand the Illumina protocol seems to produce libraries sized 250-500bp (no gel-based size selection). I was wondering what insert sizes people used for their capture, and if anyone has discovered which sizes capture most efficiently? Agilent seem to be changing their minds about what the optimal sizing is, which is concerning... I'd rather not go through a trial and error process for this as it is quite expensive and time consuming...
Any help or suggestions you can give would be hugely appreciated!
Thanks.