A robust protocol to increase NimbleGen SeqCap EZ multiplexing capacity to 96 samples

PLoS One. 2015 Apr 14;10(4):e0123872. doi: 10.1371/journal.pone.0123872. eCollection 2015.

Abstract

Contemporary genetic studies frequently involve sequencing of a targeted gene panel, for instance consisting of a set of genes associated with a specific disease. The NimbleGen SeqCap EZ Choice kit is commonly used for the targeted enrichment of sequencing libraries comprising a target size up to 7 Mb. A major drawback of this commercially available method is the exclusive use of single-indexing, meaning that at most 24 samples can be multiplexed in a single reaction. In case of relatively small target sizes, this will lead to excessive amounts of data per sample. We present an extended version of the NimbleGen SeqCap EZ protocol which allows to robustly multiplex up to 96 samples. We achieved this by incorporating Illumina dual-indexing based custom adapters into the original protocol. To further extend the optimization of cost-efficient sequencing of custom target panels, we studied the effect of higher pre-enrichment pooling factors and show that pre-enrichment pooling of up to 12 samples does not affect the quality of the data. To facilitate evaluation of capture efficiency in custom design panels, we also provide a detailed reporting tool.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / chemistry
  • DNA / metabolism
  • Genotype
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Polymorphism, Single Nucleotide
  • Sequence Analysis, DNA / methods*

Substances

  • DNA

Grants and funding

This work was funded by the Belgian National Fund for Scientific Research-Flanders (FWO) to GV and RFK and the Special Research Fund of the University of Antwerp [Bijzonder Onderzoeksfonds (BOF-IWT)] to IMvdW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.