454 gs flx

Amplification of the variable region V1–V2 of the bacterial 16S rDNA gene was utilized to assess gut microbial diversity. Primers used were 8F-338R (5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-TGCTGCCTCCCGTAGGAGT-3′) for multiplex Roche 454 GS FLX pyrosequencing. Primer design was carried out according to the manufacturer’s instructions. Initial PCR from each DNA was performed four times [20 (link),21 (link)]. After PCR, the resulting product was checked for size and purity on an agarose-Sybr safe DNA gel stain (Invitrogen, San Diego, CA, USA). The amplicons were purified using a Pure Link kit (Invitrogen, San Diego, CA, USA) and quantified using Qubit and Bioanalyzer. The pool of amplicons were mixed equimolar (four amplicons for cockroach specie) and then prepared for 454-pyrosequencing according to the manufacturer. Cycling conditions were 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 56°C for 40 s, 68°C for 40 s, and a final extension step at 68°C for 6 min.

DNA was extracted from different tissues using a slightly modified version of Aljanabi & Martinez (1997) (link) salting out method. Namely, after addition of the saline solution, the mixture was centrifuged for 30 min at 10,000 g. Also, DNA precipitation was achieved by incubation at −20 °C with 600 µL isopropanol for 30 min. Then, after washing out the pellet with 70% ethanol, we centrifuged at 10,000 g for 10 min.
For the preparation of the SSR enriched library we followed the protocol of Santana et al. (2009) (link). In brief, we started with 20 µg of high molecular weight genomic DNA (gDNA) to enrich for SSR with ISSR-PCR method (Zietkiewicz, Rafalski & Labuda, 1994 (link)), but without using the final stage of cloning in a bacterial vector. The primers used were: ISSR1 (5′-DDB (GTC) 5–3′), ISSR2 (5′-DHB (ATC) 5-3′), ISSR3 (5′YHY (GT) 5G-3′), ISSR4 (5′-HVH (CAT) 5-3′), ISSR5 (5′-NDB (TGT) 7C-3′), ISSR6 (5′-NDV (CT) 8–3′), and ISSR7 (5′-HBDB (AAC) 4–3′). For each trial microsatellite-enriched DNA we analysed in 5 µg in the Pyrosequencing platform Roche 454 GS-FLX. For each sample, a single-lane sequencing run using portioned sections of the PicoTiterPlate was performed according to the manufacturer’s protocol preparation. Sample preparation and analytical processing such as base calling, were also performed on site according to the manufacturer’s protocol.

Genome sequencing was performed by The Gene Pool genomic facility in The University of Edinburgh using Roche 454 GS-FLX and Solexa/Illumina 35-bp paired-end sequencing on standard libraries constructed according to the manufacturers instructions. Reads were assembled using Newbler v2 (Roche) and Velvet v.0.7 [52 (link)], combined using minimus2 and mapped to the reference genome of C. pecorum E58 [24 (link)] to generate 13, 10 and 9 contigs for P787, W73 and PV3056/3 respectively. In total, 12,926,259 (PV3056/3), 8,169,259 (W73) and 10,039,539 (P787) reads obtained from Solexa/Illumina sequencing and 95,683 (PV3056/3), 101,405 (W73) and 65,050 (P787) reads from Roche 454 GS-FLX sequencing were obtained. Following quality filtering, sequencing reads were mapped to the reference genome providing approximately 253× (PV3056/3), 136× (W73) and 59.4× (P787) sequencing coverage. Regions spanning the contig ends were PCR-amplified using Phusion High-fidelity DNA polymerase (NEB) and the sequence determined ensuring that each base was covered by sequence in each direction.

Pestalotiopsis fici (W106-1/CGMCC3.15140) was sequenced using a whole-genome shotgun sequencing approach at the Chinese National Human Genome Center (Shanghai, China). Three runs of Roche 454 GS FLX standard pyrosequencing generated 2,999,862 reads (a 24.5-fold sequence depth). The reads were first assembled using Newbler software Version 2.3, which produced 586 contigs. Then a DNA library of 3-kb inserts was constructed and sequenced on an Illumina/Solexa Genome analyzer using a paired-end module to construct the scaffolds. SSPACE and GapFiller software was conducted to further fill the gap and generate scaffolds. The data has been deposited at DDBJ/EMBL/GenBank under accession: ARNU00000000.

cDNA pools were amplified with the GO Taq DNA polymerase (Promega) using the 16S V4–V6 primers shown in Table S9 and suitable barcode sequences and purified using PCR purification and Gel Extraction kits (QIAGEN). PCR products were sequenced by Beckman Genomics (Grenoble, France) using the Roche 454 GS FLX+ and standard procedures. The resulting FASTA files were filtered to a minimum read length of 250 bp using Galaxy[157] (link) and blasted against the NCBI 16SMicrobial database using BLAST+ and prfectBLAST[158] (link) with standard blastn algorithm settings and 10 maximum target sequences. Further analysis was performed using MEGAN4[159] (link).

Integration sites were amplified by Linker Mediated PCR (LM-PCR), as described [27] (link). Briefly, genomic DNA was extracted from 0.5−5×10⁶ transposed cells and digested with MseI and XhoI enzyme to prevent amplification from internal mutated IR fragments. An MseI double-stranded linker was then ligated and LM-PCR performed with nested primers specific for the linker and SA IR/DR (Table S1).
LM-PCR derived amplicons were run on a Roche/454 GS FLX using titanium chemistries by GATC Biotech AG Next Gen Lab. A valid integration contained: the TAGpSAIR nested primer and the entire SA IR/DR sequence up to a TA dinucleotide.

Genomic DNA of G. sinense was sequenced using the Roche 454 GS FLX (Roche, USA) and Illumina HiSeq 2000 (Illumina, USA) NGS platforms. The sequencing process followed the manufacturer’s recommendations. Sequencing reads from Roche 454 were used to construct the primary assembly using CABOG58 (link) and then scaffolded with Illumina paired-end reads using SSPACE version 1.1. The assembly was error checked and manually corrected to build the finished scaffolds. The final scaffolds were constructed to build the chromosome-wide pseudomolecules by optical mapping.