Metagenomic Sequencing

Introduction

Sequencing of metagenomic, large-insert clone libraries facilitates the assessment of microbial communities that can be difficult to maintain in the laboratory. One benefit of using large-insert clones for metagenomic research is the ability to efficiently assemble genomic regions associated with higher-order bacterial phenomena, such as nutrient fixation and natural product biosynthesis. It remains challenging, however, to sequence large metagenomic clone libraries without redundantly oversampling maintenance vectors, which are required for propagation in a bacterial storage host. To increase sequencing efficiency, one must isolate the cloned insert from each vector backbone, at massive scale. Traditional size selection via gel electrophoresis is the best solution for this hurdle, but automated options that scale with the sequencing and bioinformatics aspects of this application are required.

Coastal® Genomics and HAMILTON Robotics developed the NIMBUS Select with Ranger® Technology for the purpose of high throughput, fully automated gel electrophoresis. The system has preparative (i.e. size selection) and quality control (i.e. analytical) capabilities. Users can size select up to 96 metagenomic clone libraries on the NIMBUS Select in a single run. Coastal® Genomics and Radiant Genomics collaborated to validate the use of the NIMBUS Select for this application.

Experiment

The NIMBUS Select recovered inserts (between 15kbp and 35kbp) while rejecting cosmid vectors (8kbp) from eight restriction enzyme-digested metagenomic clone libraries. We generated sequencing libraries with the material size selected by the NIMBUS Select (insert only) as well as non-size selected samples held back from processing (cosmid vector and insert). A Nextera™ DNA library preparation kit converted all samples into sequencing libraries, which were normalized/pooled and  sequenced on the Illumina® MiSeq platform with 2x300bp v.3 sequencing chemistry.

BBMap identified biologically irrelevant sequences by aligning raw reads to the cosmid vector sequence. SPades 3.5 then assembled the non-cosmid aligning insert reads in a de novo fashion.

Results

Analysis shows that samples processed with the NIMBUS Select platform have a greater than 10-fold reduction in cosmid vector carryover than non-size selected samples. The complexity of the assembly process is minimized in the size selected samples, resulting in a contiguity increase approximately 5-fold in magnitude when compared to non-size selected samples.

 

Metagenomic_Sequencing_Figure Metrics on cosmid contamination and assembly contiguity for samples processed by the NIMBUS Select (Insert-Only) and standard, non-size selected libraries (Cosmid+Insert).

Metagenomic_Sequencing_Table

These increases in efficiency directly translate into superior assembly statistics achieved for roughly half of the cost and in less time than competing methods. This workflow also saves significant full time employee resources when scaled over a large collection of samples.

Discussion

The NIMBUS Select with Ranger Technology assists sample preparation for BAC/cosmid library sequencing by:

  • Eliminating redundant/oversampled sequences
  • Offering turnkey automation that requires no user intervention after run initiation
  • Processing of up to 192 large-insert samples in as little as 8 hours