Beckman, K. B. | Search bioRxiv

bioRxiv2018

Measuring Illumina Size Bias Using REcount: A Novel Method for Highly Accurate Quantification of Engineered Genetic Constructs

Quantification of DNA sequence tags associated with engineered genetic constructs underlies many genomics measurements. Typically, such measurements are done using PCR to enrich sequence tags and add adapters, followed by next-generation sequencing (NGS). However, PCR amplification can introduce significant quantitative error into these measurements. Here we describe REcount, a novel PCR-free direct counting method for NGS-based quantification of engineered genetic constructs. By comparing measurements of defined plasmid pools to droplet digital PCR data, we demonstrate that this method is highly accurate and reproducible. We further demonstrate that the REcount approach is amenable to multiplexing through the use of orthogonal restriction enzymes. Finally, we use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencing platforms.

genomics↗

bioRxiv2018

Evaluating the information content of shallow shotgun metagenomics

Although microbial communities are associated with many aspects of human, environmental, plant, and animal health, there exists no cost-effective method for precisely characterizing species and genes present in such communities. While deep whole-genome shotgun (WGS) sequencing provides the highest-level of taxonomic and functional resolution, it is often prohibitively expensive for large-scale studies. The prevailing alternative, high-throughput 16S rRNA gene amplicon sequencing (16S), often does not resolve taxonomy past the genus level and provides only moderately accurate predictions of the functional profile; thus, there is currently no widely accepted approach to affordable, high-resolution, taxonomic and functional microbiome analysis. To address this technology gap, we evaluated the information content of shallow shotgun sequencing with as low as 0.5 million sequences per sample as an alternative to 16S sequencing for large human microbiome studies. We describe a library preparation protocol enabling shallow shotgun sequencing at approximately the same per-sample cost as 16S. We analyzed multiple real and simulated biological data sets, including two novel human stool samples with ultra-deep sequencing of 2.5 billion sequences per sample, and found that shallow shotgun recovers accurate species-level taxonomic and functional profiles of the human microbiome. We recognize and discuss some of the inherent limitations of shallow shotgun sequencing, and note that 16S sequencing remains a valuable and important method for taxonomic profiling of novel environments. Although deep WGS remains the gold standard for high-resolution microbiome analysis, we recommend that researchers consider shallow shotgun sequencing as a useful alternative to 16S for large-scale human microbiome research studies.

genomics↗