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Suppression Subtractive Hybridisation Allows Selective Sampling of Metagenomic Subsets of Interest.
Chew YV, Holmes AJ.
School of Molecular and Microbial Biosciences, University of Sydney, Australia.
Metagenomic studies bypass the requirement of a pure culture for analysis, focusing instead on the genetic information present in a given sample. Metagenomics have been applied to various studies, with objectives ranging from genome reconstruction, gene prospecting and ecology. However, the use of metagenomics in comparative studies has been constrained by sequencing costs and computational limitations. Efforts are underway to improve current sequencing methods and reduce the expense involved. We suggest an alternative approach - pretreatment of the sample of interest to enrich for desired subsets prior to deep sequencing. In this study, we tested the use of suppression subtractive hybridisation (SSH) for in vitro separation of metagenomic samples based on temporal variance. Faecal samples were taken from pigs at different timepoints and extracted DNA was whole genome-amplified using multiple displacement amplification (MDA). A sample collected at 31 days of age was designated the tester while a 24 day sample was denoted the driver. Following hybridisation and subtraction, tester-specific sequences are expected to be enriched in the final sample while driver-specific and common sequences are removed. Using denaturing gel gradient electrophoresis (DGGE), we found that driver-specific bands were completely removed from all final profiles while an average of 70% of common bands were successfully subtracted. Final profiles retained an average of 70% of tester-specific sequences and new sequences contributed an average of 36% of the band mobilities found in the final profiles. Tester-unique sequences were inferred to make up 78% of the final profile after SSH. We expect that using subtractive hybridisation for separation of metagenomic samples into desired subsets will provide a more effective and targeted approach to comparative studies.
Chew YV, Holmes AJ.
School of Molecular and Microbial Biosciences, University of Sydney, Australia.
Metagenomic studies bypass the requirement of a pure culture for analysis, focusing instead on the genetic information present in a given sample. Metagenomics have been applied to various studies, with objectives ranging from genome reconstruction, gene prospecting and ecology. However, the use of metagenomics in comparative studies has been constrained by sequencing costs and computational limitations. Efforts are underway to improve current sequencing methods and reduce the expense involved. We suggest an alternative approach - pretreatment of the sample of interest to enrich for desired subsets prior to deep sequencing. In this study, we tested the use of suppression subtractive hybridisation (SSH) for in vitro separation of metagenomic samples based on temporal variance. Faecal samples were taken from pigs at different timepoints and extracted DNA was whole genome-amplified using multiple displacement amplification (MDA). A sample collected at 31 days of age was designated the tester while a 24 day sample was denoted the driver. Following hybridisation and subtraction, tester-specific sequences are expected to be enriched in the final sample while driver-specific and common sequences are removed. Using denaturing gel gradient electrophoresis (DGGE), we found that driver-specific bands were completely removed from all final profiles while an average of 70% of common bands were successfully subtracted. Final profiles retained an average of 70% of tester-specific sequences and new sequences contributed an average of 36% of the band mobilities found in the final profiles. Tester-unique sequences were inferred to make up 78% of the final profile after SSH. We expect that using subtractive hybridisation for separation of metagenomic samples into desired subsets will provide a more effective and targeted approach to comparative studies.