454 gs junior

Amplicon sequencing was performed according to the manufacturer’s instructions. PCR was done using Expand High Fidelity DNA polymerase (Roche Applied Science, Basel, Switzerland). The purified PCR products were used in direct population Sanger sequencing (ABI 3730, Applied Biosystems, Foster City, USA) and UDPS (Roche/454 GS Junior, Branford, USA). The PCR amplicons were sequenced by forward direction on the Roche 454 GS Junior platform. Equimolar pooling of the DNA molecular for each patient was performed and followed by emulsion PCR and pyrosequencing on a 454 GS Junior sequencer.

Rumen microbial diversity was assessed by amplicon sequencing of bacteria using a 454 GS Junior (Roche) at Luke (formerly known as MTT Agrifood Research Finland). Archaea, ciliate protozoa and fungi diversity were evaluated using MiSeq (Illumina) platforms. The change in sequencing platform was related to the rapid development of Illumina sequencing capacity at the time when sequencing was done and lower costs in relation to Roche 454. Primer sequences used for amplification are described in S1 Table. For bacteria, five 10 bp barcodes (Roche TCB No. 005–2009) were added at the 5’-end of both forward and reverse primers to make amplicon pooling possible. The PCR amplification, library preparation and sequencing on a 454 GS Junior (Roche) were performed following standard procedures (S1 Text). Sequencing of archaeal libraries was performed at Fasteris SA (Geneva, Switzerland) as described in [23 (link)]. Ciliate protozoa and anaerobic fungi libraries were constructed and sequenced at LGC Genomics (Berlin, Germany) following the 250 bp paired-end protocol.

In 8 coral Acropora samples and 1 seawater sample collected from Kochi, regions V1–V2 of the bacterial 16S rRNA gene were amplified by PCR using the bacterial universal primers 27F and 341R (5′-CTGCTGCCTCCCGTAGG-3′). DNA tagging PCR was used to fuse unique tags to each PCR product, which was conducted as described (2 (link)). Amplicons from the 9 Kochi samples were quantified and pooled in equal amounts. Multiplex sequencing was performed with a Roche 454 GS junior with Titanium chemistry - System (Roche 454 Life Sciences, Branford, CT, USA) at Mission Biotech (Taipei, Taiwan).
Raw sequencing reads were sorted into samples according to barcodes using an in-house sorter script (http://140.109.29.21/scripts/). After sorting and trimming with data from specific primers, high-quality reads were extracted using MOTHUR (52 (link)) with the following criteria: 1) read lengths between 280 and 350 bp; 2) average quality score >20; 3) homopolymer length <8 bp; and 4) removal of reads with any ambiguous base (N). Thereafter, the 4 nucleotide tags and primer sequences were removed. Chimeric reads were inspected and eliminated by UCHIME (15 (link)) with USEARCH v7.0.1090 (parameters: reference mode, rdp_gold database, and mindiv of 3). A total of 91,211 qualified sequences were retained for subsequent analyses.