The 81st Annual Meeting of the American Association of Physical Anthropologists (2012)


Optimizing enrichment strategies for next-generation sequencing using ancient TB

KELLY M. HARKINS1, MEAGAN A. RUBEL1,2, LUZ-ANDREA PFISTER1 and ANNE C. STONE1.

1School of Human Evolution and Social Change, Arizona State University, 2Department of Anthropology, University of Pennsylvania

Friday All day, Plaza Level Add to calendar

The characterization of strains of ancient Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), allows us to reconstruct phylogenetic relationships critical to understanding the dynamics of TB interactions with humans prior to and after the Age of Exploration. While previous studies have successfully extracted ancient TB (Ariazza et al. 1995; Braun et al. 1998; Salo et al. 1994), the recovery of ancient pathogen DNA remains problematic due to degradation processes and contamination. Recent advancements in enrichment strategies coupled with next-generation sequencing have been successful in isolating, amplifying and sequencing ancient DNA (e.g. Burbano et al. 2010; Maricic et al. 2010; Stiller et al. 2009). The ability to detect M. tuberculosis in ancient samples, target genomic regions of interest, and create a high quality DNA library are crucial steps in this process. Here we report on a number of analyses that: 1) Test the sensitivity of newly designed qPCR assays for detecting multi-copy insertion elements in M. tuberculosis, 2) Test the Direct-Multiplex Sequencing method (Stiller et. al 2009) for targeted capture of 100+ phylogentically-informative SNPs (Hershberg et al. 2008); and, 3) Create and measure the quality of the enriched DNA libraries designed for next-generation sequencing, as well as various library purification kits. Preliminary results indicate that our IS1081 qPCR assay is sensitive in low quantity DNA, but that small multiplex PCR products are lost with SPRI purification methods during library construction. Further research will continue to optimize these steps, increasing the effectiveness of sequencing efforts.

This research was funded by the National Science Foundation grants #BCS-0612222 and BCS-1063939.

Tweet
comments powered by Disqus