Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform

MSystems
Johanna B HolmJacques Ravel

Abstract

Amplification, sequencing, and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300-bp paired-end reads of higher quality than those produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-step PCR amplification protocol is also described that allows for targeting of different amplicon regions, and enhances amplification success from samples with low bacterial bioburden. IMPORTANCE Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high-throughput sequencing have made it a widely adopted approach, especially for projects that necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here pro...Continue Reading

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Citations

May 26, 2020·Applied Microbiology and Biotechnology·Leena MalayilAmy R Sapkota
Oct 2, 2019·The Journal of Infectious Diseases·Jeanne TamarelleJacques Ravel
Sep 10, 2020·Frontiers in Cellular and Infection Microbiology·Supriya D MehtaRobert C Bailey
Aug 22, 2020·The Journal of Infectious Diseases·Supriya D MehtaFredrick Otieno
Feb 4, 2021·International Journal of STD & AIDS·Supriya D MehtaRupert Kaul
Feb 9, 2020·Clinical Nutrition : Official Journal of the European Society of Parenteral and Enteral Nutrition·Rupak ShivakotiRebecca M Brotman
Feb 26, 2021·PeerJ·Liying LowUNKNOWN West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT)
Apr 30, 2021·JAMA Network Open·Chiaowen Joyce HsiaoNauder Faraday
May 13, 2021·Applied Microbiology and Biotechnology·Suhana ChattopadhyayAmy R Sapkota
May 18, 2021·Frontiers in Cellular and Infection Microbiology·Anne L DunlopElizabeth J Corwin
May 26, 2021·Scientific Reports·Remy VilletteMartin Larsen
Apr 28, 2021·The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences·Michelle ShardellRebecca M Brotman
Jul 17, 2021·Frontiers in Cellular and Infection Microbiology·Sarah E BrownRebecca M Brotman
Sep 10, 2021·Sexually Transmitted Infections·Juliano NovakCamila Marconi
Oct 5, 2021·Frontiers in Pharmacology·Xiaoying HouFengguo Xu

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Datasets Mentioned

BETA
PRJNA489669

Methods Mentioned

BETA
PCR
Illumina sequencing
electrophoresis
DNA assay

Software Mentioned

BLASTn
split
MiSeq
BLAST
R Package
TagCleaner
HiSeq Control
ggplot2
R package irr
R

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