Hi all.
We performed our first amplicon titanium run this week using the Lib-L emPCR kit. I’ve attached the Agilent DNA chip (standard not high sensitivity) of this amplicon pool to this post. My thoughts were that although the fragments were larger than we would see with our gDNA libraries it still looked within range of and should amplify well during emPCR.
emPCR went well, 2 LV cups produced 6.7 million enriched beads. 2 million were then loaded per region on a 2 region plate. The run was analyzed using the “signal processing for amplicon” pipeline.
The run yield was very disappointing, only 15.63% passing filter. 72.5 % of the failures were due to short, this being split 50/50 between short quality and short primer. I can accept that some of the reads would be lost to short quality but I can understand why we lost so many reads to short primer when the initial Agilent trace seemed to suggest that there were no short fragments present in the pool.
Has anyone seen similar with titanium amplicon experiments? And has anyone sequenced fragments this large?
We performed our first amplicon titanium run this week using the Lib-L emPCR kit. I’ve attached the Agilent DNA chip (standard not high sensitivity) of this amplicon pool to this post. My thoughts were that although the fragments were larger than we would see with our gDNA libraries it still looked within range of and should amplify well during emPCR.
emPCR went well, 2 LV cups produced 6.7 million enriched beads. 2 million were then loaded per region on a 2 region plate. The run was analyzed using the “signal processing for amplicon” pipeline.
The run yield was very disappointing, only 15.63% passing filter. 72.5 % of the failures were due to short, this being split 50/50 between short quality and short primer. I can accept that some of the reads would be lost to short quality but I can understand why we lost so many reads to short primer when the initial Agilent trace seemed to suggest that there were no short fragments present in the pool.
Has anyone seen similar with titanium amplicon experiments? And has anyone sequenced fragments this large?
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