The fixed AS samples were washed by 0.85% NaCl twice (by centrifugation at 10,000 g for 5 min) and the samples on membrane were detached by vortexing for 1 min after adding 0.85% NaCl and 1-mm acid-washed glass beads. After collecting the biomass by centrifugation, DNA (including AS, biomass on membranes and isolates) was extracted by using the FastDNA SPIN kit for Soil as described before27 (link). All the extracts were quantified by a spectrophotometer (NanoDrop-1000, Thermo, USA) and visualized by agarose gel electrophoresis.
To obtain the community-level mycobacterial rpoB amplicon, primers and PCR condition were referred to the ref.25 (link). The 16 samples were multiplexed by the different 6-nt barcodes adding to the 5′ end of the forward or reverse primer28 (link). In addition, the adaptor A and B for 454 pyrosequencing were added at the 5′-end of the barcoded forward primers and reverse primer, respectively. The PCR products were pooled at equal mass after purification. The rpoB amplicon pool was sent to Genome Research Center in the University of Hong Kong to perform 454 pyrosequencing (Titanium platform, Roche, USA). Genomic DNA of the four mycobacterial isolates was sent to Beijing Genomic Institute (Shenzhen, China) for Illumina sequencing on the Hiseq2000 platform with inserted length of 800 bp and paired-end (PE) reading strategy.
To obtain the community-level mycobacterial rpoB amplicon, primers and PCR condition were referred to the ref.25 (link). The 16 samples were multiplexed by the different 6-nt barcodes adding to the 5′ end of the forward or reverse primer28 (link). In addition, the adaptor A and B for 454 pyrosequencing were added at the 5′-end of the barcoded forward primers and reverse primer, respectively. The PCR products were pooled at equal mass after purification. The rpoB amplicon pool was sent to Genome Research Center in the University of Hong Kong to perform 454 pyrosequencing (Titanium platform, Roche, USA). Genomic DNA of the four mycobacterial isolates was sent to Beijing Genomic Institute (Shenzhen, China) for Illumina sequencing on the Hiseq2000 platform with inserted length of 800 bp and paired-end (PE) reading strategy.
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