Jun 15, 2016

Adapterama I: Universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)

BioRxiv : the Preprint Server for Biology
Travis C. GlennBrant C. Faircloth

Abstract

Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing costs of: 1) start-up (i.e., doing NGS for the first time); 2) buy-in (i.e., getting the smallest possible amount of data from a run); and 3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduc...Continue Reading

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Mentioned in this Paper

Computer Software
DNA Restriction
DNA Repair
Primer Extension
Restriction Site
Research Personnel
Nucleic Acid Sequencing
Sequencing
Massively-Parallel Sequencing
Oligonucleotide Primers

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