PT  - JOURNAL ARTICLE
AU  - Jiahao Ni
AU  - Jiao Pan
AU  - Yaohai Wang
AU  - Tianhao Chen
AU  - Xinshi Feng
AU  - Yichen Li
AU  - Tongtong Lin
AU  - Michael Lynch
AU  - Hongan Long
AU  - Weiyi Li
TI  - One-Step PCR Amplicon Library Construction (OSPALC, version 1)
AID  - 10.1101/2022.10.26.511284
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.10.26.511284
4099  - http://biorxiv.org/content/early/2022/10/27/2022.10.26.511284.short
4100  - http://biorxiv.org/content/early/2022/10/27/2022.10.26.511284.full
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.