Abstract
BackgroundMore extensive use of metagenomic shotgun sequencing in microbiome research relies on the development of high-throughput, cost-effective sequencing. Here we present a comprehensive evaluation of the performance of the new high-throughput sequencing platform BGISEQ-500 for metagenomic shotgun sequencing and compare its performance with that of 2 Illumina platforms.FindingsUsing fecal samples from 20 healthy individuals, we evaluated the intra-platform reproducibility for metagenomic sequencing on the BGISEQ-500 platform in a setup comprising 8 library replicates and 8 sequencing replicates. Cross-platform consistency was evaluated by comparing 20 pairwise replicates on the BGISEQ-500 platform vs the Illumina HiSeq 2000 platform and the Illumina HiSeq 4000 platform. In addition, we compared the performance of the 2 Illumina platforms against each other. By a newly developed overall accuracy quality control method, an average of 82.45 million high-quality reads (96.06% of raw reads) per sample, with 90.56% of bases scoring Q30 and above, was obtained using the BGISEQ-500 platform. Quantitative analyses revealed extremely high reproducibility between BGISEQ-500 intra-platform replicates. Cross-platform replicates differed slightly more than intra-platform replicates, yet a high consistency was observed. Only a low percentage (2.02%–3.25%) of genes exhibited significant differences in relative abundance comparing the BGISEQ-500 and HiSeq platforms, with a bias toward genes with higher GC content being enriched on the HiSeq platforms.ConclusionsOur study provides the first set of performance metrics for human gut metagenomic sequencing data using BGISEQ-500. The high accuracy and technical reproducibility confirm the applicability of the new platform for metagenomic studies, though caution is still warranted when combining metagenomic data from different platforms.
Highlights
MethodsTwenty Swedish healthy adults living in the Stockholm regional area were enrolled as part of a large study cohort: “Characterization of the intestinal microbiome in patients with IgA deficiency.” The detailed inclusion and exclusion criteria were as follows: (1) no diagnosed gastrointestinal problems (inflammatory bowel disease, celiac disease, or lactose intolerance); (2) no antibiotic treatment for at least 60 days; (3) no intake of yoghurt products for at least 5 days prior to sampling
More extensive use of metagenomic shotgun sequencing in microbiome research relies on the development of high-throughput, cost-effective sequencing
We present a comprehensive evaluation of the performance of the new high-throughput sequencing platform BGISEQ-500 for metagenomic shotgun sequencing and compare its performance with that of 2 Illumina platforms
Summary
Twenty Swedish healthy adults living in the Stockholm regional area were enrolled as part of a large study cohort: “Characterization of the intestinal microbiome in patients with IgA deficiency.” The detailed inclusion and exclusion criteria were as follows: (1) no diagnosed gastrointestinal problems (inflammatory bowel disease, celiac disease, or lactose intolerance); (2) no antibiotic treatment for at least 60 days; (3) no intake of yoghurt products for at least 5 days prior to sampling. To assess the consistency of relative abundance identification of gut microbial genes, we performed Spearman correlation analysis based on highly reproducible (HR) genes and species profiles (Supplementary Methods) Both sequence replicates and library replicates showed high consistency at the gene level (Spearman’s rho > 0.91) and species level (Spearman’s rho > 0.97) (Fig. 2B). Paired tests of gene abundances revealed no significant difference between BGISEQ-500 technical replicates (false discovery rate [FDR] < 0.05, Benjamini-Hochberg adjustment) These findings demonstrate that the BGISEQ-500 platform, across the entire process of library preparation and sequencing of metagenomic DNA samples, provides highly reproducible and well-controllable results. To further document the quantitative consistency and performance regarding GC content observed for the BGISEQ-500 and HiSeq 2000 platforms, the same 20 DNA samples were processed to construct libraries and sequenced on an Illumina HiSeq 4000 platform using the HiSeq 3000/4000SBS Kit (300 cycles) for 100 bp of paired-end reads. The high crossplatform quantitative consistency was further documented using a HiSeq 4000 dataset, with a lower number of genes exhibiting differences in abundance between the platforms, and exhibiting the same bimodal distribution pattern of GC content of these genes
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