Improved high-molecular-weight DNA extraction, nanopore sequencing and metagenomic assembly from the human gut microbiome

Abstract

Short-read metagenomic sequencing and de novo genome assembly of the human gut microbiome can yield draft bacterial genomes without isolation and culture. However, bacterial genomes assembled from short-read sequencing are often fragmented. Furthermore, these metagenome-assembled genomes often exclude repeated genomic elements, such as mobile genetic elements, compromising our understanding of the contribution of these elements to important bacterial phenotypes. Although long-read sequencing has been applied successfully to the assembly of contiguous bacterial isolate genomes, extraction of DNA of sufficient molecular weight, purity and quantity for metagenomic sequencing from stool samples can be challenging. Here, we present a protocol for the extraction of microgram quantities of high-molecular-weight DNA from human stool samples that are suitable for downstream long-read sequencing applications. We also present Lathe ( www.github.com/bhattlab/lathe ), a computational workflow for long-read basecalling, assembly, consensus refinement with long reads or Illumina short reads and genome circularization. Altogether, this protocol can yield high-quality contiguous or circular bacterial genomes from a complex human gut sample in approximately 10 d, with 2 d of hands-on bench and computational effort.

Fig. 1 |. High-molecular-weight DNA extraction workflow.

Frozen stool is best divided into aliquots by using a biopsy punch with a plunger, taking extreme caution with the blade. After dividing the stool into aliquots, the DNA extraction workflow proceeds with sample suspension, enzymatic and chemical lysis and DNA purification. The Qiagen Genomic-tip is a column-based purification method designed to minimize shearing of DNA. Allow all reagents to flow through the Genomic-tip column by gravity; do not use a syringe or vacuum to pull liquid through, as this will destroy the column. After DNA has been extracted, large fragments can then be selected for using SPRI beads prepared in a custom buffer. Extracted DNA should be assessed for concentration, contamination and size distribution by using a Qubit fluorometer, NanoDrop spectrophotometer and Agilent TapeStation, respectively (not depicted). After DNA has been extracted and meets quality thresholds, it can be carried through library preparation protocols for nanopore sequencing. EtOH, ethanol.

Fig. 2 |. Post-sequencing bioinformatic workflow.

After sequencing, the computational assembly workflow (Step 25) is used to perform basecalling of raw nanopore signal before performing assembly by using either Flye or Canu assemblers. The workflow then provides optional polishing steps by aligning short or long reads back to the assembly and correcting single nucleotide errors and indels (green) with consensus bases (orange) from the aligned reads. Finally, the workflow is used to perform circularization steps through self-alignment and trimming or assembly of endpoint contigs.