6 research outputs found
International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant
This study tested the claim that
digital PCR (dPCR) can offer highly reproducible quantitative measurements
in disparate laboratories. Twenty-one laboratories measured four blinded
samples containing different quantities of a <i>KRAS</i> fragment encoding G12D, an important genetic marker for guiding
therapy of certain cancers. This marker is challenging to quantify
reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence
of competing wild type sequences and the need for calibration. Using
dPCR, 18 laboratories were able to quantify the G12D marker within
12% of each other in all samples. Three laboratories appeared to measure
consistently outlying results; however, proper application of a follow-up
analysis recommendation rectified their data. Our findings show that
dPCR has demonstrable reproducibility across a large number of laboratories
without calibration. This could enable the reproducible application
of molecular stratification to guide therapy and, potentially, for
molecular diagnostics
Accurate whole human genome sequencing using reversible terminator chemistry
DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications