Abstract
BackgroundCurrent library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols.ResultsExtensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM.ConclusionsA sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.
Highlights
Current library preparation protocols for Illumina Illumina high-throughput sequencer (HiSeq) and MiSeq Deoxyribonucleic acid (DNA) sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells
Current Illumina protocols for sample loading onto the flow cells call for ≥2 nM denatured cDNA libraries
2–4 nM libraries are first denatured by adding an equal volume of NaOH (0.2 N or 0.1 N, respectively, in MiSeq or HiSeq)
Summary
Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has not been reported. Most high throughput DNA sequencing facilities use Illumina sequencers such as HiSeq or MiSeq. Current Illumina protocols for sample loading onto the flow cells call for ≥2 nM denatured cDNA libraries. At 50–100 mM, NaOH effectively denatures double-strand cDNA to form single-strand
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