Efficient method/approach for heterozygote calling in sequence reads?

Colin Dolphin colin.dolphin at kcl.ac.uk
Tue Jun 27 04:41:06 EST 2000


We are identifying sequence variation in a gene of interest by firstly
using DHPLC to  identify those PCR products originating from individuals
who are heterozygous at, at least, one position. We do not have access to
an onsite "core" sequencing facility so have had to use a sequencing
service. After some deliberation we opted for one using a Licor/IR
because we were assured that the Licor system/chemistry was superior in
identifying - and scoring - heterozygote positions. Even though the
original PCR product (which has to be free of non-specific products for
the DHPLC) is reamplified, using nested primers tailed with M13 fwd or
rev sequences, and then this product sequenced the resulting sequence
data has repeatedly failed to score known heterozygous positions on both
fwd and rev reads. Thus we cannot be sure that it is also identifying
"unknown" heterozygous positions.

Can someone help/advise with the best way to use this system to
accurately and unambiguously score such heterozygous positions in PCR
products or, alterbatively, share their experiences of heterozygote
calling in sequencing reads?

Colin

Colin Dolphin
Dept of Pharmacy
Kings College London
London SE1 8WA
colin.dolphin at kcl.ac.uk






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