You’re invited to join our webinarHow library preparation affects sequencing accuracy
Advances in next-generation DNA sequencing have allowed the detection of cell-specific genetic variation, revealing mutations that affect a wide range of phenotypes and pathologies, including cancer. Somatic cancer variants are often found at low allelic frequency due to tumor heterogeneity and/or contamination by normal cells. However, detecting low frequency variation (1 – 5%) in mixed genetic populations is challenging, and requires careful consideration of potential sources of error. Typical sample preparation methods can induce artifactual errors in otherwise fresh, high-quality DNA samples. Polymerase misincorporation during PCR can also generate errors that ultimately appear in sequencing data. These errors confound the detection of true low-frequency mutations that are critical to understanding the genetic diversity of tumors. In this webinar, we will discuss how library preparation affects sequencing accuracy and variant calling.
In Part 1, Laurence Ettwiller will discuss a recent Science paper from NEB, which describes how sequencing errors caused by DNA damage has affected large-scale genomics studies. It is estimated that 41% of the sequencing runs from the 1000 Genomes Project and 73% of the runs addressed in The Cancer Genome Atlas show signs of DNA damage. More specifically, one-third or more of G-to-T mutations in certain reads may be false positives. Understanding the extent of DNA damage, and its contribution to sequencing errors, can help researchers identify true genetic variation from errors associated with sequencing and library preparation artifacts.
In Part 2, Jennifer Ong will present data from a recent publication that describes an accurate PCR polymerase fidelity assay based on PacBio Single-Molecule Real-Time sequencing. This assay was used to examine diverse sources of PCR errors, including polymerase base substitution errors, chimeric product formation from template-switching, and DNA damage from thermocycling. The contribution of PCR errors to next-generation library preparation will also be discussed.
Date: Wednesday May 31, 2017
Time: 2:00pm EST
|Laurence Etwillier, Ph.D. Staff Scientist
New England Biolabs
|Jennifer Ong, Ph.D.
New England Biolabs