Metagenomic extractions typically involve an entire biome lysis, with subsequent nucleic acid isolation and purification from complex, mechanically diverse, and thus difficult-to-lyse matrices. The most common starting materials in metagenomic extractions are soil and fecal samples; these samples are the most difficult to standardize in terms of processing procedures.
Challenges in processing result from variables such as complex matrices with a wide range of mechanical and rheological properties and the inhabitance of innate PCR inhibitors and degrading enzymes. Additionally, the large diversity of cells, tissue remains, and microorganisms produce genomic extractions of varying nucleic acid length and molecular weight.
Fecal samples come in seven basic forms, as classified per the Bristol Stool Chart (Figure 1), and can range from the turdaceous hard solid matrices (Type 1) to a thixotropic liquid consistency (Type 7).1 Fecal cellular material types range from the soft-shelled, easily lysed E. coli to echinococcal oocytes and other parasitic ova, some of the most difficult samples to lyse, as well as solid food remains.
In recent years, several general protocols for stool homogenization and nucleic acid purification have been devised, and there are several articles where they were compared in depth.2 A common motif for the protocols included mechanical lysis of the stool sample, wherein the complete, quantitative lysis of all of the stool constituents was achieved, with even the most resistant single-cell constituents, such as gram-positive bacterial spores and ova, releasing their nucleic acids into a protective buffer.
Mechanical lysis was followed by pre-washing involving some form of flocculation and/or other separation steps to remove lignic materials and flocculate inhibitors. The final step included standard protocols for nucleic acid extraction, most commonly solid-phase bind-wash-elute procedures.
This report will review one of the more popular protocols, based on the application of mechanical sample lysis followed up with flocculation and high-capacity liquid silica slurry binding and solid-phase extraction, the combination of which provides the highest yield and purity of the DNA from all types of fecal materials.
Soil samples present equally difficult challenges in sample preparation and nucleic acid extraction due to many of the same variables enumerated in this article, including complexity of matrix types as well as diversity of biological materials contained, and can also have added natural inhibitors in the form of hard mineral matrices and different content and ratios of humics, fulvics, polyphenols, and polysaccharides.