Pregledni rad
Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy
Mitchell M. Holland
; Forensic Science Program, The Pennsylvania State University, University Park, Pa, USA
Megan R. McQuillan
; Oakland County Sheriff’s Office Forensic Laboratory, Pontiac, Mich, USA
Katherine A. O’Hanlon
; Forensic Science Program, The Pennsylvania State University, University Park, Pa, USA
Sažetak
Aim To use parallel array pyrosequencing to deconvolute
mixtures of mitochondrial DNA (mtDNA) sequence and
provide high resolution analysis of mtDNA heteroplasmy.
Methods The hypervariable segment 1 (HV1) of the mtDNA
control region was analyzed from 30 individuals using
the 454 GS Junior instrument. Mock mixtures were used
to evaluate the system’s ability to deconvolute mixtures
and to reliably detect heteroplasmy, including heteroplasmic
differences between 5 family members of the same
maternal lineage. Amplicon sequencing was performed
on polymerase chain reaction (PCR) products generated
with primers that included multiplex identifiers (MID) and
adaptors for pyrosequencing. Data analysis was performed
using NextGENe® software. The analysis of an autosomal
short tandem repeat (STR) locus (D18S51) and a Y-STR locus
(DYS389 I/II) was performed simultaneously with a portion
of HV1 to illustrate that multiplexing can encompass
different markers of forensic interest.
Results Mixtures, including heteroplasmic variants, can be
detected routinely down to a component ratio of 1:250 (20
minor variant copies with a coverage rate of 5000 sequences)
and can be readily detected down to 1:1000 (0.1%) with
expanded coverage. Amplicon sequences from D18S51,
DYS389 I/II, and the second half of HV1 were successfully
partitioned and analyzed.
Conclusions The ability to routinely deconvolute mtDNA
mixtures down to a level of 1:250 allows for high resolution
analysis of mtDNA heteroplasmy, and for differentiation
of individuals from the same maternal lineage. The
pyrosequencing approach results in poor resolution of
homopolymeric sequences, and PCR/sequencing artifacts
require a filtering mechanism similar to that for STR stutter
and spectral bleed through. In addition, chimeric sequences
from jumping PCR must be addressed to make
the method operational.
Ključne riječi
Hrčak ID:
71440
URI
Datum izdavanja:
15.6.2011.
Posjeta: 1.699 *