Qiaxtractor

Viral RNA was extracted using the QIAxtractor (Qiagen, Hilden, Germany) platform. Extracted RNA was denatured at 97°C for 5 minutes and reverse transcription-PCR was performed using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with random hexamers (Pharmacia Biotech, Uppsala, Norway) by following a single thermal cycle of 25°C for 5 minutes, 37°C for 60 minutes and 95°C for 5 minutes [24 (link)].
5μl of the cDNA was used for G-typing and P-typing PCR reactions of each specimen, following previously established procedures for eight G (1–4, 8–10, 12) types and six P (4, 6, 8–11) types rotavirus strains [25 (link)–26 (link)].

Genomic DNA was extracted using a QIAxtractor (Qiagen), and library preparation was performed according to the Illumina protocol. Index-tagged libraries were created, and 96 isolates multiplexed per lane and sequenced using the Illumina HiSeq 2000 platform (Illumina) to generate 100-bp paired-end reads. The average sequencing depth was 77-fold, with a minimum of 48-fold.

Each swab was suspended in 400 µl sterile phosphate buffered saline (PBS) after thawing at room temperature. DNA was extracted from the swab/PBS suspension using an adapted whole blood protocol on the QIAxtractor (Qiagen, Crawley, UK) automated instrument and eluted into a final volume of 50 µl DX Elution Buffer (Qiagen).

Bacterial genomic DNA was extracted using the QIAxtractor (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Library preparation was conducted according to the Illumina protocol and sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA, USA) with 100-cycle paired-end runs. Sequence data were retrieved for a further 1,517 open access E. coli isolates associated with bloodstream infections (17 (link), 18 (link)). Of these, 424 were isolated between January 2006 and December 2012 at the Cambridge University Hospitals NHS Foundation Trust, and 1,093 were submitted to the British Society for Antimicrobial Chemotherapy Bacteraemia Resistance Surveillance Project by 11 UK hospitals between 2001 and 2011 (for details, see www. bsacsurv.org and Table S1 in the supplemental material) (31 (link)). Previous description and analysis of these genomes (17 (link), 18 (link)) did not include comparisons with isolates from livestock or meat.

DNA extraction was performed with the QIAxtractor (Qiagen) instrument according to the manufacturer’s instructions. Illumina sequencing libraries with a 450-bp insert size were prepared according to the manufacturer’s protocols and sequenced on an Illumina HiSeq2000 with paired-end reads with a length of 100 bp. Ninety-six samples were multiplexed per lane to give an average depth of coverage of ~90-fold. We assembled paired-end sequence reads by employing an assembly and improvement pipeline (51 ) that is based on Velvet (52 (link)) and subsequently annotated the de novo assemblies with Prokka (53 (link)). To perform the pangenome analysis, we took the output from Prokka and analyzed it with Roary (54 (link)).

Each tracheal swab obtained from the in vivo co-infection study was serially diluted (ten-fold) in growth media and used to inoculate sub-confluent (80–90% confluency) LMH monolayers in 6-well plates. After 1 hour of adsorption at 37°C, the monolayer was covered with methyl-cellulose overlay media (1% w/v methyl-cellulose in DMEM, with 10% FBS, 50 μg/mL ampicillin, 50 μg/mL gentamicin) and incubated at 37°C. After 24 to 48 hours of incubation, 20 isolated plaques from each swab sample were carefully picked using a micropipette and an inverted light microscope. Then each plaque was individually propagated by inoculating of LMH monolayers grown in 12-well plates. Three plaque picking purification rounds, with one freeze/thaw cycle in-between each round, was performed before a final amplification in one well of a 6-well plate. Nucleic acid was extracted from 200 μL of plaque-purified virus using the automated QIAxtractor (Qiagen) system and Vx reagents (Qiagen). DNA was eluted in 100 μL of elution buffer. In order to control for any recombination that could occur in vitro during the process of inoculation and plaque purification, pure CSW-1 and V1-99 stocks were mixed and immediately used to co-inoculate additional cultures of LMH cells using 1:1 ratio of 1×10⁴ PFU of each virus. Isolated plaques were then picked and processed as described above.