Best Practices for Illumina Library Preparation

Curr Protoc Hum Genet. 2019 Jun;102(1):e86. doi: 10.1002/cphg.86.

Abstract

In this unit, we describe a set of protocols and recommendations for Illumina library preparation. We review best practices in template quantitation methods; template fragmentation methodologies; solid-phase reverse-immobilization cleanup, including buffer exchange and size selection; end repair, A-tailing, and adapter ligation; indexing strategies; considerations regarding whether to use polymerase chain reaction; final library quantification methodologies; and normalization and pooling strategies. These workflows are applicable to both high-throughput and low-throughput Illumina library preparation and should help reduce bias, increase cost effectiveness, and produce high library yields. This is an extensive update of the previous version of this unit. © 2019 by John Wiley & Sons, Inc.

Keywords: Illumina; PCR; bias; indexing; library; next-generation sequencing; sample preparation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • DNA Fragmentation
  • DNA Transposable Elements
  • Fluorescent Dyes / chemistry
  • Gene Library*
  • High-Throughput Nucleotide Sequencing / methods*
  • Polymerase Chain Reaction / methods
  • Templates, Genetic

Substances

  • DNA Transposable Elements
  • Fluorescent Dyes