RT Journal Article
SR Electronic
T1 One-Step PCR Amplicon Library Construction (OSPALC, version 1)
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.10.26.511284
DO 10.1101/2022.10.26.511284
A1 Jiahao Ni
A1 Jiao Pan
A1 Yaohai Wang
A1 Tianhao Chen
A1 Xinshi Feng
A1 Yichen Li
A1 Tongtong Lin
A1 Michael Lynch
A1 Hongan Long
A1 Weiyi Li
YR 2022
UL http://biorxiv.org/content/early/2022/10/27/2022.10.26.511284.abstract
AB High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. However, commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. Here, to overcome this technical hurdle, we present a one-step PCR amplicon library construction (OSPALC) protocol for amplicon library preparations in the lab along with a QIIME2-based amplicon analysis pipeline. High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library construction is completed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 1/15 of the typical cost via service providers. Empirically tested primers to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Criteria to design primers targeting at any regions of prokaryotic and eukaryotic genomes are also suggested. In principle, OSPALC can be conveniently used in amplicon library constructions of any target gene of both prokaryotes and eukaryotes at the DNA or RNA levels, and will facilitate research in numerous fields.Competing Interest StatementThe authors have declared no competing interest.