Library preparation for metagenomic sequencing with Illumina

Krasnenko AYu1, Eliseev AYu2, Borisevich DI1, Tsukanov KYu1, Davydova AI1, Il’inskii VV1
About authors

1 Genotek Inc., Moscow, Russia

2 Moscow South-West High School No. 1543, Moscow, Russia

Correspondence should be addressed: Anna Krasnenko
Nastavnicheskiy per. 17, str. 1, pod. 14, Moscow, 105120; moc.liamg@oknensarkanna

About paper

Acknowledgements: the authors thank Daria Plakhina and Ivan Stetsenok of Genotek for their help and Sergey Glagolev of Moscow South-West High School No. 1543 for his valuable advice and comments.

Contribution of authors to this work: Krasnenko AYu, Eliseev AYu — analysis of literature, research planning and implementation, data analysis and interpretation; Borisevich DI, Tsukanov KYu — bioinformatic analysis; Davydova AI — drafting of a manuscript; Ilinsky VV — research planning, scientific advisor. All authors participated in editing of the manuscript.

Received: 2017-04-12 Accepted: 2017-04-24

Metagenomic sequencing is widely used in both scientific research and clinical practice for characterization of taxonomic profiles including estimation of relative abundance of prokaryotes in microbial communities in various media. Metagenomic sequencing of single marker genes is an excellent tool for studying the human microbiome. Unlike whole-genome sequencing, it targets those genome regions that can be instrumental in identification of microorganism species and genus. The 16S ribosomal RNA (16S rRNA) gene sequence is highly conserved but at the same time there are regions containing species- specific sequences that can discriminate between different bacteria and archaea. These regions can be amplified using universal primers, which makes the whole procedure more cost-effective and less time-consuming. Good primers and protocol design for PCR at the step of library preparation is crucial for achieving high data accuracy. Below we describe how to choose the optimal PCR protocol and universal primers to amplify V3 and V4 regions of the 16S rRNA gene for further sample sequencing using Illumnia platform.

Keywords: microbiome, metagenomic sequencing, ribosomal RNA, 16S rRNA, polymerase chain reaction, universal primers, library preparation, double barcode