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A specific combination of dual index adaptors decreases the sensitivity of amplicon sequencing with the Illumina platform.

ABSTRACT: Amplicon sequencing is a powerful approach in microbiome studies as it detects live organisms with high sensitivity. This approach determines the composition of sequence variants of marker genes using high-throughput DNA sequencers. The use of dual index adaptors is the fundamental technique for pooling DNA libraries for Illumina sequencers and is believed not to affect the results. However, here, we observed a decrease of sequence quality in samples containing a specific combination of indexes, namely N704 and S507 in Nextera kits, in multiple runs on the Illumina MiSeq sequencer operated in different facilities. This decrease was also observed when sequencing randomly fragmented DNA of Escherichia coli and was not observed when either individual adaptor was used. Each end of the DNA library with this index combination contains a complementary sequence motif, which potentially inhibits proper cluster generation and/or subsequent sequencing. Community analysis of the 16S and 18S rRNA amplicons using QIIME2 revealed significant decreases in ?-diversity in the samples containing the N704/S507 index combination, resulting from loss of low-abundance sequence variants during denoising. Our data underscore the importance of quality validation of sequence reads in developing dual index techniques and suggest cautious interpretation of microbiome data containing low-quality sequence reads.

SUBMITTER: Hirose Y

PROVIDER: S-EPMC7547650 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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A specific combination of dual index adaptors decreases the sensitivity of amplicon sequencing with the Illumina platform.

Hirose Yuu Y Shiozaki Takuhei T Hamano Itsuki I Yoshihara Shizue S Tokumoto Hayato H Eki Toshihiko T Harada Naomi N

DNA research : an international journal for rapid publication of reports on genes and genomes 20200801 4

Amplicon sequencing is a powerful approach in microbiome studies as it detects live organisms with high sensitivity. This approach determines the composition of sequence variants of marker genes using high-throughput DNA sequencers. The use of dual index adaptors is the fundamental technique for pooling DNA libraries for Illumina sequencers and is believed not to affect the results. However, here, we observed a decrease of sequence quality in samples containing a specific combination of indexes, ...[more]

PMID: 32810209

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Project description:Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized.We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms.The library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost.Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.

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A specific combination of dual index adaptors decreases the sensitivity of amplicon sequencing with the Illumina platform.

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A specific combination of dual index adaptors decreases the sensitivity of amplicon sequencing with the Illumina platform.

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