Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform
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...
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American Society for Microbiology
2019
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oai:doaj.org-article:d6a13bab938f4f0faae5f0daf99212e12021-12-02T19:47:34ZUltrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform10.1128/mSystems.00029-192379-5077https://doaj.org/article/d6a13bab938f4f0faae5f0daf99212e12019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00029-19https://doaj.org/toc/2379-5077ABSTRACT 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 provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method.Johanna B. HolmMichael S. HumphrysCourtney K. RobinsonMatthew L. SettlesSandra OttLi FuHongqiu YangPawel GajerXin HeElias McCombPatti E. GravittKhalil G. GhanemRebecca M. BrotmanJacques RavelAmerican Society for Microbiologyarticle16S RNAamplicon sequencingPCRultrahigh throughputMicrobiologyQR1-502ENmSystems, Vol 4, Iss 1 (2019) |
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DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
16S RNA amplicon sequencing PCR ultrahigh throughput Microbiology QR1-502 |
| spellingShingle |
16S RNA amplicon sequencing PCR ultrahigh throughput Microbiology QR1-502 Johanna B. Holm Michael S. Humphrys Courtney K. Robinson Matthew L. Settles Sandra Ott Li Fu Hongqiu Yang Pawel Gajer Xin He Elias McComb Patti E. Gravitt Khalil G. Ghanem Rebecca M. Brotman Jacques Ravel Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform |
| description |
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 provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method. |
| format |
article |
| author |
Johanna B. Holm Michael S. Humphrys Courtney K. Robinson Matthew L. Settles Sandra Ott Li Fu Hongqiu Yang Pawel Gajer Xin He Elias McComb Patti E. Gravitt Khalil G. Ghanem Rebecca M. Brotman Jacques Ravel |
| author_facet |
Johanna B. Holm Michael S. Humphrys Courtney K. Robinson Matthew L. Settles Sandra Ott Li Fu Hongqiu Yang Pawel Gajer Xin He Elias McComb Patti E. Gravitt Khalil G. Ghanem Rebecca M. Brotman Jacques Ravel |
| author_sort |
Johanna B. Holm |
| title |
Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform |
| title_short |
Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform |
| title_full |
Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform |
| title_fullStr |
Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform |
| title_full_unstemmed |
Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform |
| title_sort |
ultrahigh-throughput multiplexing and sequencing of >500-base-pair amplicon regions on the illumina hiseq 2500 platform |
| publisher |
American Society for Microbiology |
| publishDate |
2019 |
| url |
https://doaj.org/article/d6a13bab938f4f0faae5f0daf99212e1 |
| work_keys_str_mv |
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1718375992553111552 |