The Extreme Microbiome Project is testing various methods for recovering DNA and RNA from our unique samples, as well as evaluating bioinformatics tools to detect and describe sample components.
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The Extreme Microbiome Project is testing various methods for recovering DNA and RNA from our unique samples, as well as evaluating bioinformatics tools to detect and describe sample components.
The Metagenomics Research Group is currently finalizing DNA extraction protocols to determine which method is most effective for the unique samples we are collecting.
The following protocols are currently being tested:
– CTAB (Omega) with enzymatic digestion and sapphire beater beats
– Phenol
– MoBio
The Metagenomics Research Group is currently finalizing RNA extraction protocols to determine which method is most effective for the unique samples we are collecting.
The following protocols are currently being tested:
– Trizol LS with Diamond and Sapphire abrasive beater beads
– Hot Phenol
The XMP uses multiple sequencing approaches in an effort to reduce bias and correctly establish genus and species-level identifications.
Illumina: Sequencing is done with paired end 250×250 or greater
PacBio: Site specific sequencing will also be performed using the PacBio platform with the latest chemistry
Oxford Nanopore: Site specific sequencing is being done using nanopore technologies and the MinION
Life Technologies: Ion Torrent will be used with 400 base pair reads
The MGRG is now benchmarking bioinformatics tools available for metagenomic analysis with the goal of identifying and developing the most reliable methods for studying microbiomes and metagenomics.
Major tools we are using for taxa characterization include:
We will also align our samples to specific sequences (strains, virulent plasmids, genes linked to antibiotic resistance, etc.) using the Burrows Wheeler Aligner (BWA) to address clinically relevant questions regarding the virulence and pathogenic elements of microbiomes.
Although tools are growing more sophisticated and databases encompassing a greater variety of sequences, all methods remain flawed. Our team of bioinformatics researchers will evaluate the accuracy of the available tools using MGRG positive controls.
The Metagenomics Research Group of the Association of Biomolecular Resource Facilities has developed three metagenomics reference standards that are now available for purchase. These contain six to ten strains with in an even or staggered mix and can be used for many applications. Strains were selected in collaboration with NIST and ATCC and are Class I BCL 1, having highly varying GC content, Gram reaction and Order.
The three available standards can be found here: ABRF-MGRG Metagenomics Reference Standards
The ABRF MGRG, in collaboration with Sigma Aldrich, is developing the MAC4L Polyzyme mix for digestion of cell walls from the range of species present in metagenomic samples.
This mixture includes:
Protocols using MPZ: