Dataset Information

Comparative evaluation of a new magnetic bead-based DNA extraction method from fecal samples for downstream next-generation 16S rRNA gene sequencing.

ABSTRACT: We are colonized by a vast population of genetically diverse microbes, the majority of which are unculturable bacteria that reside within the gastrointestinal tract. As affordable, advanced next-generation sequencing technologies become more widely available, important discoveries about the composition and function of these microbes become increasingly possible. In addition to rapid advancement in sequencing technologies, automated systems have been developed for nucleic acid extraction; however, these methods have yet to be widely used for the isolation of bacterial DNA from fecal samples. Here, we adapted Promega's Maxwell® RSC PureFood GMO and Authentication kit for use with fecal samples and compared it to the commonly used Qiagen QIAamp® PowerFecal® kit. Results showed that the two approaches yielded similar measures of DNA purity and successful next-generation sequencing amplification and produced comparable composition of microbial communities. However, DNA extraction with the Maxwell® RSC kit produced higher concentrations with a lower fecal sample input weight and took a fraction of the time compared to the QIAamp® PowerFecal® protocol. The results of this study demonstrate that the Promega Maxwell® RSC system can be used for medium-throughput DNA extraction in a time-efficient manner without compromising the quality of the downstream sequencing.

SUBMITTER: McGaughey KD

PROVIDER: S-EPMC6107275 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Comparative evaluation of a new magnetic bead-based DNA extraction method from fecal samples for downstream next-generation 16S rRNA gene sequencing.

McGaughey Kara D KD Yilmaz-Swenson Tulay T Elsayed Nourhan M NM Cruz Dianne A DA Rodriguez Ramona R RR Kritzer Michael D MD Peterchev Angel V AV Gray Megan M Lewis Samantha R SR Roach Jeffrey J Wetsel William C WC Williamson Douglas E DE

PloS one 20180823 8

We are colonized by a vast population of genetically diverse microbes, the majority of which are unculturable bacteria that reside within the gastrointestinal tract. As affordable, advanced next-generation sequencing technologies become more widely available, important discoveries about the composition and function of these microbes become increasingly possible. In addition to rapid advancement in sequencing technologies, automated systems have been developed for nucleic acid extraction; however ...[more]

PMID: 30138447

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Project description:Microbial community analysis based on the 16S rRNA-gene is used to investigate both beneficial and harmful microorganisms in various fields and environments. Recently, the next-generation sequencing (NGS) technology has enabled rapid and accurate microbial community analysis. Despite these advantages of NGS based metagenomics study, sample transport, storage conditions, amplification, library preparation kits, sequencing, and bioinformatics procedures can bias microbial community analysis results. In this study, eight mock communities were pooled from genomic DNA of Lactobacillus acidophilus KCTC 3164T, Limosilactobacillus fermentum KCTC 3112T, Lactobacillus gasseri KCTC 3163T, Lacticaseibacillus paracasei subsp. paracasei KCTC 3510T, Limosilactobacillus reuteri KCTC 3594T, Lactococcus lactis subsp. lactis KCTC 3769T, Bifidobacterium animalis subsp. lactis KCTC 5854T, and Bifidobacterium breve KCTC 3220T. The genomic DNAs were quantified by droplet digital PCR (ddPCR) and were mixed as mock communities. The mock communities were amplified with various 16S rRNA gene universal primer pairs and sequenced by MiSeq, IonTorrent, MGIseq-2000, Sequel II, and MinION NGS platforms. In a comparison of primer-dependent bias, the microbial profiles of V1-V2 and V3 regions were similar to the original ratio of the mock communities, while the microbial profiles of the V1-V3 region were relatively biased. In a comparison of platform-dependent bias, the sequence read from short-read platforms (MiSeq, IonTorrent, and MGIseq-2000) showed lower bias than that of long-read platforms (Sequel II and MinION). Meanwhile, the sequences read from Sequel II and MinION platforms were relatively biased in some mock communities. In the data of all NGS platforms and regions, L. acidophilus was greatly underrepresented while Lactococcus lactis subsp. lactis was generally overrepresented. In all samples of this study, the bias index (BI) was calculated and PCA was performed for comparison. The samples with biased relative abundance showed high BI values and were separated in the PCA results. In particular, analysis of regions rich in AT and GC poses problems for genome assembly, which can lead to sequencing bias. According to this comparative analysis, the development of reference material (RM) material has been proposed to calibrate the bias in microbiome analysis.

Dataset Information

Comparative evaluation of a new magnetic bead-based DNA extraction method from fecal samples for downstream next-generation 16S rRNA gene sequencing.

Publications

Comparative evaluation of a new magnetic bead-based DNA extraction method from fecal samples for downstream next-generation 16S rRNA gene sequencing.

Similar Datasets

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