Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing

Abstract

The large amount of DNA sequence data generated by high-throughput sequencing technologies often allows multiple samples to be sequenced in parallel on a single sequencing run. This is particularly true if subsets of the genome are studied rather than complete genomes. In recent years, target capture from sequencing libraries has largely replaced polymerase chain reaction (PCR) as the preferred method of target enrichment. Parallelizing target capture and sequencing for multiple samples requires the incorporation of sample-specific barcodes into sequencing libraries, which is necessary to trace back the sample source of each sequence. This protocol describes a fast and reliable method for the preparation of barcoded ("indexed") sequencing libraries for Illumina's Genome Analyzer platform. The protocol avoids expensive commercial library preparation kits and can be performed in a 96-well plate setup using multi-channel pipettes, requiring not more than two or three days of lab work. Libraries can be prepared from any type of double-stranded DNA, even if present in subnanogram quantity.

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus

“…Shotgun Illumina libraries were constructed using previously developed protocols (Meyer and Kircher 2010), with minor modifications. In brief, for each sample, 100 ng of DNA (or 30 μl of sample extract for low-yield dentine and control samples) was constructed into indexed libraries using the NEBNext DNA Library Prep Master set for 454 (E6070; New England Biolabs) according to manufacturer instructions, except that SPRI bead purification was replaced with silica adsorption (Qiagen MinElute PCR Purification kit).…”

“…In brief, for each sample, 100 ng of DNA (or 30 μl of sample extract for low-yield dentine and control samples) was constructed into indexed libraries using the NEBNext DNA Library Prep Master set for 454 (E6070; New England Biolabs) according to manufacturer instructions, except that SPRI bead purification was replaced with silica adsorption (Qiagen MinElute PCR Purification kit). Blunt end adapters (IS1/IS3 and IS2/IS3) were prepared following Meyer and Kircher (2010) and used for ligation at a concentration of 0.5 μM in a final volume of 50 μl. The Bst Polymerase fill-in reaction was inactivated after incubation by heating to 80°C for 20 minutes and then freezing overnight.…”

High-throughput sequencing of complete human mtDNA genomes from the Caucasus and West Asia: high diversity and demographic inferences

“…About 400 ng of DNA was used for tagging the nebulized PCR product for each individual with a specific tag sequence, as described elsewhere, 19 and Illumina Genome Analyzer II (Illumina Inc., San Diego, CA, USA) libraries were prepared according to the protocol described elsewhere. 20 Fifty individuals with unique tags were pooled in each library in equimolar ratios. Subsequently, libraries were sequenced with 36 and/or 76-bp reads in one lane of an Illumina flow cell (Cluster Generation kit V2, FC-103-300Â sequencing chemistry, San Diego, CA, USA) according to the manufacturer's instructions.…”

“…21 Raw sequences called from Ibis were separated by sample using their index read, allowing one mismatch and the loss of the first base. 20 Reads were then filtered for sequence quality and complexity. In this step, reads having 45 bases with a quality score below 10 (PHRED scale) and reads with sequence entropy (calculated by summing -p*log2(p) for each of the four nucleotides) below 1.0 were removed.…”

Complete mtDNA genomes of Filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the Asia-Pacific region

“…DNA sequencing and sequence assembly DNA libraries were prepared using a multiplex method developed for the Illumina Genome Analyzer (GA) platform, 16 coupled with a target-enrichment method specific for human mtDNA. 17 DNA libraries were sequenced on an Illumina GA IIx machine (Illumina Inc., San Diego, CA, USA) with post processing using Illumina software followed by the Improved Base Identification System.…”