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Old 12-08-2014, 03:36 PM   #1
Location: Los Angeles, CA

Join Date: Dec 2014
Posts: 30
Default AmpliSeq using NextSeq 2x75bp PE sequencing

I am considering using Life's AmpliSeq protocol and modifying it to use Illumina indices for sequencing on a NextSeq. In FFPE mode, AmpliSeq generates 150bp amplicons. However, I believe these are tiled and it looks like there is less than 2X overlap. If I sequence using 2x75bp sequencing, would my targets be completely covered?

My concern is that there are "partially digested primers" between the adapters and the target sequence. If the amplicon is sequenced for 75bp from both ends, then that will leave a gap in the middle.
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Last edited by Ingeneious; 12-08-2014 at 03:40 PM.
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Old 12-08-2014, 08:33 PM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,234

NextSeq 150 sequencing kit has reagents for 175 cycles. You may do more cycles than 2x75 if your indices are not 25 nt.
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