# Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

## TL;DR

The paper introduces a new method called METa assembly that allows functional metagenomic libraries to be prepared from very small DNA samples, enabling the discovery of antibiotic resistance genes.

## Contribution

The novel contribution is a library preparation method requiring only 30 ng of DNA, enabling functional metagenomics from low-biomass samples.

## Key findings

- METa assembly can prepare functional libraries from as little as 30.5 ng of DNA.
- The method captured a novel streptothricin acetyltransferase gene and tetracycline resistance transporters.
- Functional metagenomic libraries were successfully prepared from aquatic and fecal swab samples.

## Abstract

A significant challenge in the field of microbiology is the functional annotation of novel genes from microbiomes. The increasing pace of sequencing technology development has made solving this challenge in a high-throughput manner even more important. Functional metagenomics offers a sequence-naive and cultivation-independent solution. Unfortunately, most methods for constructing functional metagenomic libraries require large input masses of metagenomic DNA, putting many sample types out of reach. Here, we show that our functional metagenomic library preparation method, METa assembly, can be used to prepare useful libraries from much lower input DNA quantities. Standard methods of functional metagenomic library preparation generally call for 5–60 µg of input metagenomic DNA. We demonstrate that the threshold for input DNA mass can be lowered at least to 30.5 ng, a 3-log decrease from prior art. We prepared functional metagenomic libraries using between 30.5 ng and 100 ng of metagenomic DNA and found that despite their limited input mass, they were sufficient to link MFS transporters lacking substrate-specific annotations to tetracycline resistance and capture a gene encoding a novel GNAT family acetyltransferase that represents a new streptothricin acetyltransferase, satB. Our preparation of functional metagenomic libraries from aquatic samples and a human stool swab demonstrates that METa assembly can be used to prepare functional metagenomic libraries from microbiomes that were previously incompatible with this approach.

Bacterial genes in microbial communities, including those that give resistance to antibiotics, are often so novel that sequencing-based approaches cannot predict their functions. Functional metagenomic libraries offer a high-throughput, sequence-naive solution to this problem, but their use is often held back due to their need for large quantities of metagenomic DNA. We demonstrate that our functional metagenomic library preparation method, METa assembly, can prepare these libraries using as little as ~30 ng of DNA, approximately 1,000-fold less than other methods. We use METa assembly to prepare functional metagenomic libraries from low-biomass aquatic and fecal swab microbiomes and show that they are home to novel tetracycline efflux pumps and a new family of streptothricin resistance gene, respectively. The efficiency of the METa assembly library preparation method makes many otherwise off-limits, low-biomass microbiome samples compatible with functional metagenomics.