Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics

Abstract

Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.

Functional meta-omics provide critical insights into long- and short-read assemblies

“…Besides software-driven differences, e.g. assembler choice, the extent of the differences in the metagenomic reconstruction approaches will also depend on associated costs and the thus achievable sequencing depth [ 21 ], complexity/composition of the sample and other factors, such as DNA extraction approaches [ 22 ], or library preparation methods [ 23 ], especially for low-abundance organisms. In addition to our newly generated human-borne multi-omic data (GDB), we used publicly available SR and LR metagenomic data originating from the same respective sample (Zymo, NWC or Rumen).…”

Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams

“…In recent years, several research groups ( Besemer et al., 2012 ; Ren et al., 2017 ; Ren, Gao & Elser, 2017 ; Dancer, Shears & Platt, 1997 ) have successfully used kit-based methods for DNA extraction and subsequent 16S ribosomal RNA gene amplicon sequencing on GFS samples. However, the requirements for whole genome shotgun sequencing currently include at least 50 ng of input DNA to minimize bias due to PCR reactions during library preparation ( Kebschull & Zador, 2015 ; Bower et al., 2015 ; Thomas, Gilbert & Meyer, 2012 ; Chafee, Maignien & Simmons, 2015 ; Peng et al., 2020 ). Here, we address the utility and efficiency of the “gold” standard phenol-chloroform extraction ( Dairawan & Shett, 2020 ), and three alternative methodologies to identify the process(es) that yield not only the highest quantity but also quality of DNA, from GFS sediments.…”

“…The advent of high-throughput sequencing technologies has brought hitherto inconceivable capacities to characterize the microbial ecology of both well-studied (Jansson & Hofmockel, 2018;Nielsen & Ji, 2015) and under-explored environments (Hotaling, Hood & Hamilton, samples. However, the requirements for whole genome shotgun sequencing currently include at least 50 ng of input DNA to minimize bias due to PCR reactions during library preparation (Kebschull & Zador, 2015;Bower et al, 2015;Thomas, Gilbert & Meyer, 2012;Chafee, Maignien & Simmons, 2015;Peng et al, 2020). Here, we address the utility and efficiency of the ''gold'' standard phenol-chloroform extraction (Dairawan & Shett, 2020), and three alternative methodologies to identify the process(es) that yield not only the highest quantity but also quality of DNA, from GFS sediments.…”

Peer Review #3 of "Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams (v0.2)"

“…In recent years, several research groups (Besemer et al 2012;Dancer, Shears, and Platt 1997) have successfully used kit-based methods for DNA extraction and subsequent 16S ribosomal RNA gene amplicon sequencing on GFS samples. However, the requirements for whole genome shotgun sequencing currently include at least 50 ng of input DNA to minimize bias due to PCR reactions during library preparation (Kebschull and Zador 2015;Bowers et al 2015;Thomas, Gilbert, and Meyer 2012;Chafee, Maignien, and Simmons 2015;Peng et al 2020). Here, we address the utility and efficiency of the "gold" standard phenol-chloroform extraction (Dairawan and Shetty 2020), and three alternative methodologies to identify the process(es) that yield not only the highest quantity but also quality of DNA, from GFS sediments.…”