DNA was isolated as previously described (Kumar et al., 2011b (link)) and multiplexed bacterial tag-encoded FLX 16S pyrosequencing was performed using the Titanium platform (Roche Applied Science, Indianapolis, IN, USA). Two regions of the 16S rRNA genes were sequenced: V1–V3 (spanning E.coli 16S gene regions 8–27 and 519–536) and V7–V9 (spanning E.coli 16S gene regions 1099–1114 and 1528–1541). The primers used for sequencing have been previously described (Kumar et al., 2011b (link)). Each primer is capable of detecting a range of genera that the other fails to recover. Together they allow the recovery of a wider range of the microbiome than is possible with a single primer alone. However, some genera are picked up by both primers. Thus, to prevent over counting, the number of sequences assigned to an OTU by both primers was reduced by half. Primer averaging was carried out as previously described (Kumar et al., 2011b (link)) using the implementation in the PhyloTOAST software suite (Dabdoub et al., 2016 (link)). Sequences with an average quality score of 30 over a sliding window of 50 bp and length >200 bp were assigned a taxonomic identity by alignment to the HOMD database (Chen et al., 2010 (link)) using the Blastn algorithm. Analyses were conducted using the QIIME (Caporaso et al., 2010 (link)) and PhyloToAST (Dabdoub et al., 2016 (link)) pipelines.
Titanium platform
Manufactured by Roche
Sourced in United States
The Titanium platform is a versatile laboratory equipment designed for various analytical applications. It offers reliable and consistent performance in scientific research and testing. The core function of the Titanium platform is to provide a stable and precise instrument for data collection and analysis, supporting the needs of research and development professionals.
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4 protocols using titanium platform
1
Bacterial 16S Sequencing and Microbiome Analysis
2
Metagenomic Fosmid Library Sequencing
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A total of 3,300 randomly selected fosmid clones were sequenced on one full lane of the 454 GS-FLX Genome Sequencer System using the Titanium platform (Roche, Brandford, CT, USA) following the manufacturer’s protocol. Repeats in raw sequenced reads obtained were removed using RepeatMasker (http://www.repeatmasker.org ). The vector and host sequences were filtered by BLASTN, with an E-value cutoff of 1e-3. The filtered reads were assembled using the Newbler assembly software, developed by 454 Life Sciences (version 2.6, Roche). Non-overlapping fragment singletons were clustered using the CD-HIT software [58 (link)] to minimize redundant sequences. The overall process of metagenomic data preparation and analysis is summarized in Additional file 1 : Figure S1. The entire sequences of the bagasse fosmid library have been deposited to the NCBI Sequence Read Archive (SRA), which can be accessed using the accession number: SRX493840.
3
Mycobacterial Community Profiling by 454
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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.
4
Multiplex 16S rRNA Sequencing Protocol
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Multiplexed bacterial tag-encoded FLX amplicon pyrosequencing was performed using the Titanium platform (Roche Applied Science, Indianapolis, IN, USA) as previously described20 (link) in a commercial facility (MRDNALab. Shallowater, TX, USA). Briefly, a single step PCR with broad-range universal primers and 22 cycles of amplification was used to amplify the 16S rRNA genes as well as to introduce adaptor sequences and sample-specific bar-code oligonucleotide tags into the DNA. Two regions of the 16S rRNA genes were sequenced: V1–V3 and V7–V9. The primers used for sequencing have been previously described21 (link). Adaptor sequences were trimmed from raw data with 98% or more of bases demonstrating a quality control of 30 and sequences binned into individual sample collections based on bar-code sequence tags, which were then trimmed. Sequences <300 bp were discarded and the rest were clustered into species- level operational taxonomic units (s-OTUs) at 97% sequence similarity and assigned a taxonomic identity by alignment to locally hosted version of the HOMD database using the Blastn algorithm. Analyses were conducted using the QIIME pipeline22 (link), as well as our own internally developed analysis pipeline PhyloToAST23 . Results were visualized using the Python library matplotlib. Phylogenetic tree data was visualized through the Interactive Tree Of Life webserver24 (link).
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