Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics.

Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.

[Isolation and characterization of Bdellovibrio strains from the ocean].

OBJECTIVE: To isolate and identify the new Bdellovibrio strains from the sea mud of Shenzhen bay and to preliminarily study their biological characteristics. METHODS: We isolated Bdellovibrio strains by DNB(dilute nutrient broth) double-layer plate method. Their 16S rDNAs were sequenced and their morphologies were examined under electron microscope. We identified these strains according to the ninth edition of Bergey's manual of determinative bacteriology. We also studied their biological characteristics through physiological tests. RESULTS: We isolated 2 strains of Bdellovibrio sp. (5#-12 and 5#-sh06) from sea mud of Shenzhen bay. Both strains grew between 209C and 35 degrees C, with 259C and 309C as optimal temperature for 5#-12 and 5#-sh06, respectively. They grew between pH 6.1 and 8.6, and the opticCmal pH for both was 7.2. Lysis experiments on 58 strains of pathogens were conducted and the results showed that 5#-12 and 5#-sh06 lysed 46 and 48 strains, corresponding to 79.3% and 82.8% of lysis abilities. Taken both two Bdellovibrio strains together, they lysed 96.6% (56 strains) of tested pathogens and 100% of tested vibrios (39 strains). CONCLUSION: The results demonstrated that Bdellovibrio have potential and significant application prospect for elimination of pathogens.