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Metagenomics

Next-generation sequencing (NGS) of DNA extracted from metagenomic samples has enabled the identification and characterization of low abundance and unculturable bacteria within environmental samples. However, several hurdles to obtaining an accurate representation of the microbial population in these samples still exist. Bias introduced by amplification strategies, whether MDA- or PCR-based, can skew the relative representation of bacterial and viral populations within the sample. Furthermore, the relative difficulty of extracting DNA from some bacterial species can limit or exclude representation of “hard-to-crack” microbes within a population. The Accel-NGS® 1S Plus and 2S DNA Library Preparation Kits allow users to enhance the accuracy of their metagenomics analyses using strategies that avoid amplification-introduced bias and enhance recovery of hard to extract microbial DNA.

SWIFT PRODUCT LINES COMPATIBLE WITH METAGENOMICS:

Accel-NGS 1S Plus DNA Library Kit

The Swift advantage:
  • Compatible with harsh DNA extraction methods to enable sequencing of difficult samples.
  • Direct adaptation of ssDNA converts all DNA within the sample into library.

Accel-NGS 2S DNA Library Kits

The Swift advantage:
  • Coverage of extreme base compositions allows detection of AT-/GC-rich microbes in samples.
  • Efficient library preparation reduces or eliminates bias-introducing amplification methods.

Minimizing Bias in Metagenomic Sample Sequencing

PCR-free Libraries from 100 ng of an Artificial Microbial Community

Several methods for preparing DNA libraries utilize PCR, which can introduce bias against genomes with extreme base composition (AT-/GC-rich). This bias can be attributed to preferential amplification of DNA with balanced base composition by DNA polymerases. As a result, microbes with extreme base composition, like Plasmodium falciparum (19% GC) or Streptomyces avermitilis (71% GC), can be misrepresented or can even go undetected in metagenomic analyses utilizing PCR amplified libraries. Additionally, differential efficiencies among polymerases can make inter-library comparisons difficult. Therefore, PCR-free metagenomic analyses provide the most accurate and comparable diversity and abundance data.

NGS libraries were prepared from 100 ng aliquots of genomic DNA from an artificial microbial community (AMC) comprised of six different microbial genomes with an Accel-NGS 2S PCR-Free DNA Kit and a competitor’s kit requiring six cycles of PCR. As the AMC was constructed with known quantities of six different microbes, the accuracy with which the final library represents the input can be analyzed along with evenness of coverage for each of the genomes.

Accel-NGS 2S PCR-Free Provides a More Accurate Representation of the AMC

AP_Metag_Image_Accurate AMC

Accel-NGS 2S PCR-Free Provides More Even Coverage of Extreme Genomes

AP_Metag_Image_2S PF Coverage

Accel-NGS 2S PCR-Free Captures More of the AMC Genomes

AP_Metag_Image_AMC Genomes

The percent of each genome in the AMC that was covered by 1 or fewer reads, demonstrating that Accel-NGS 2S PCR-Free preserves the whole genome coverage of each microbe in the AMC, while the Competitor +PCR misses 7.6% of P. falciparum 3D7. Accel-NGS 2S PCR-Free is shown in blue, while the Competitor +PCR is shown in red.

Whole Genome Sequencing of “Hard-to-Crack” Microbes

Overcoming Low DNA Recovery with Sodium Hydroxide Boiling Lysis Extraction

Whole genome sequencing (WGS) generally requires high quality, intact, double-stranded DNA (dsDNA) in concentrations that can be hard to achieve when working with microbes due to the nature of the cell wall. In order to extract high quality DNA, most researchers use organism-specific enzymatic digestion or time-consuming bead beating methods to break down the cell wall before extraction. However, these methods can be insufficient to recover enough genomic DNA necessary for NGS library preparation. The use of sodium hydroxide boiling lysis (NaOH boiling) for DNA extraction provides increased recovery of DNA in a fraction of the time of bead beating, but denatures samples into single stranded DNA (ssDNA) that cannot be adapted by most library preparation workflows. The compatibility of the Accel-NGS 1S Plus DNA Kit with ssDNA enables analysis of microbes that are difficult to sequence by conventional methods due to insufficient DNA recovery. Combined with NaOH boiling, Accel-NGS 1S Plus permits extraction, library preparation and quantification to be completed in one day, drastically reducing the turn-around-time for microbial genomic and metagenomics projects. The coverage and sequencing data below indicate that, in addition to a fast workflow, NaOH boiling with an Accel-NGS 1S Plus Kit creates high quality libraries that exhibit diversity and evenness of coverage that is comparable to the traditional bead beating workflow.

Enhanced DNA Recovery and High Quality Libraries Using NaOH Boiling with Accel-NGS 1S Plus

AP_Metag_Table_NaOH Boiling

Superior lysing of Eggerthella sp. HGA1 Gram-positive bacteria is demonstrated by the 47-fold increase in DNA recovery by NaOH boiling. Both libraries were sequenced at a depth of 2M reads. Alignment results were compared using Picard GCmetrics and SAMtools. Both extraction methods provide high quality DNA for genome sequencing.
Accel-NGS 1S Plus Produces Sequenceable Libraries from DNA Extracted with Either Bead Beating or NaOH Boiling

AP_Metag_Image_1SPlus Bead Beat vs NaOH

Picard GC Bias metrics analysis of Eggerthella alignments after DNA extraction by bead beating or NaOH boiling. Both libraries had similar evenness of coverage across the GC content of the genome.
Enhanced DNA Recovery and Consensus Genome Assembly Using NaOH Boiling

AP_Metag_Table_NaOH Boiling_2

Superior lysing of Facklamia sp. HGF4 Gram-positive bacteria is demonstrated by the 103-fold increase in DNA recovery by NaOH boiling. Reads were assembled de novo into a 1.9 Mb consensus genome. Both extraction methods provide high quality DNA for genome sequencing.
High Diversity Libraries Regardless of Extraction Technique

AP_Metag_1SPlus-DNAExtraction

Detection of ssDNA Viruses Within Viromes

Avoiding MDA-Based Methods Improves Relative Abundance Accuracy

Single-stranded DNA (ssDNA) viruses are an understudied group, due to difficulty in detection with NGS library preparation as the single-stranded nature of these viruses cannot be directly adapted by traditional library preparation methods. The use of multiple displacement amplification (MDA) allows detection of ssDNA viruses by traditional library preparations by converting ssDNA into readily adaptable dsDNA. However, MDA also eliminates the ability to accurately determine the relative abundance of the virome in the community as rolling circle amplification amplifies some genomes more efficiently than others. The Accel-NGS 1S Plus DNA Library Preparation Kit allows users to bypass problematic MDA methods and directly adapt both ssDNA and dsDNA phages, enabling accurate detection of abundance levels. Artificial viral communities (AVCs) were constructed to demonstrate the excellent performance of Accel-NGS 1S without the need for MDA. De novo assembly of each AVC was able to fully assemble both ssDNA and dsDNA phages present in the AVC into individual contigs, demonstrating comprehensive coverage by the 1S Plus Kit.

Accel-NGS 1S Plus Accurately Reproduces Expected Distributions for ssDNA and dsDNA Phages

AP_Metag_Image_1SPlus-Detection

The similarity of the observed 1S Plus library distribution and expected distribution for each library demonstrates the accurate representation of input by 1S Plus libraries. These three AVCs contain different proportions of the ssDNA phage PhiX174 and M13, mixed with dsDNA phages (all Caudovirales).
Accel-NGS 1S Plus Enables Reliable Abundance Detection of ssDNA and dsDNA Phages

AP_Metag_Image_1S Plus_ssDNA_dsDNA

A linear regression plot of the expected abundance of each phage present compared to the observed abundance demonstrates that Accel-NGS 1S Plus libraries accurately detect the abundance of each phage (R2 = 0.92).