Copycontrol fosmid library production kit

The Northern pike fosmid genomic DNA library was constructed by Bio S&T (Québec, Canada) from high molecular weight DNA extracted from the liver of a male E. lucius from Ille et Vilaine, France, using the CopyControl Fosmid Library Production Kit with pCC1FOS vectors (Epicentre, USA) following the manufacturer’s instructions. The resulting fosmid library contained around 500,000 non-amplified clones that were arrayed in pools of 250 individual fosmids in ten 96-well plates.
Northern pike fosmid clones were screened by PCR (S5 Table) to identify individual fosmids containing amhby and amha. To sequence the two ~ 40 kb fosmids, purified fosmid DNA was first fragmented into approximately 1.5 kb fragments using the Nebulizer kit supplied in the TOPO shotgun Subcloning kit (Invitrogen, Carlsbad, CA), and then sub-cloned into pCR4Blunt-TOPO vectors. Individual plasmid DNAs were sequenced from both ends with Sanger sequencing using M13R and M13F primers. The resulting sequences were then assembled using ChromasPro version 2.1.6 (Technelysium Pty Ltd, South Brisbane, Australia).

Metagenomic DNA extraction was performed with 8 g of a pooled sample from all collected soils using the UltraClean Mega Soil DNA Kit (MOBIO Laboratories, Carlsbad, CA), with some modifications to the manufacturer's protocol. Soil samples were subjected to 60°C–65°C to assure complete lysis of microorganisms and to obtain good quality DNA. Additionally, steps involving mixing by vortex were eliminated to prevent DNA fragmentation. The extracted DNA was concentrated in 5 mol/L sodium chloride–ethanol solution, and then eluted in Tris‐EDTA. DNA samples were separated by low‐point agarose gel electrophoresis at 30V during 16 hr. A 30‐kb fragment of high molecular weight (HMW) metagenomic DNA was selected and purified using QIAquick Gel Extraction Kit (QIAGEN GmbH, Germany) as previously reported (Prakash & Taylor, 2012). CopyControl Fosmid Library Production Kit (Epicentre, Madison, WI, USA) was used to construct the metagenomic library following manufacturer's instructions, using 0.25 μg HMW DNA and 0.5 μg of vector. The obtained metagenomic library (7,296 metagenomic clones) in E. coli EPI300 was stored at −80°C in 20% (vol/vol) glycerol‐LB media with chloramphenicol until used.

Total DNA was isolated from the filter using the Metagenomic DNA Isolation Kit for Water (Epicentre Biotechnologies). The high molecular weight DNA obtained was used directly to construct a metagenomic fosmid library with the fosmid pCC1FOS, using the Copy Control Fosmid Library Production kit (Epicentre Biotechnologies), following manufacturer's instructions. The prepared library comprised approximately 11,600 clones in Escherichia coli EPI300-T1 strain. The colonies were pooled into groups of five clones and arrayed in 25 96-well microtiter plates. Colonies of the metagenomic library were manually picked with sterile pipette tips, and cultivated in 96-well plates of 2 mL per well, filling each well with 0.2 mL of LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with 12.5 mg/mL chloramphenicol. Cells from five different colonies were pooled per well. These cultures were grown at 37°C for 24 h and used for functional screening, for storage at −80°C and to inoculate new cultures for DNA preparation for sequencing.

MDV strain 814 was propagated in CEFs until 80–90% CPE was obtained. The viral DNA was purified from infected cells using hypotonic lysis to release virus particles followed by micrococcal nuclease treatment to degrade cellular DNA36 (link). An MDV fosmid library was constructed using the Copy Control Fosmid Library Production Kit (Epicentre). The viral DNA was sheared into 25~50 kb fragments. After blunt treatment and phosphorylation of the ends, the viral DNA was selected for 36–48 kb fragments with pulsed field gel electrophoresis (PFGE, Bio-Rad). The collected fragments were subsequently inserted into the cloning-ready fosmid vector pCC1FOS. The recombinant fosmids were packaged using MaxPlax Lambda Packaging Extracts and plated on EPI300-T1 plating cells (Epicentre). The presence of the DNA fragment inserts in these fosmids was confirmed by end sequencing with a pair of specific primers (5′-GGA TGT GCT GCA AGG CGA TTA AGT TGG-3′ and 5′-CTC GTA TGT TGT GTG GAA TTG TGA GC-3′).

Total DNA was extracted using the liquid nitrogen grinding method [16] (link) and finally suspended in MilliQ water. The DNA sample was further determined in 1% (w/v) agarose gels, and NanoDrop measurements gave a concentration of 350 ng/µl with A₂₆₀/A₂₈₀ of 1.90. A total of 5 µg of total DNA was pyrosequenced using Roche 454 GS FLX system (Majorbio, China). Since the length of generated reads were long enough to annotate (90% reads >400 base pairs), assembly of the raw sequences was not performed. The unassembled metagenomic dataset was subjected to further analysis. At the same time, a metagenomic library was constructed using total DNA and CopyControl™ Fosmid Library Production Kit (Epicentre, Madison, WI) according to the manufacture's protocol. The collection of the metagenomic library contained total of 7,776 large-insert fosmid clones.

Metagenomic DNA was extracted from 200 μL of SRF, SAB and LAB using the BIO101 FastDNA® Spin Kit for Soil (Qbiogene, Cambridge, UK) following the supplier’s protocol but with 3 × 30-s bead beating with 1-min intervals on ice. The libraries were constructed using the CopyControl™ pCC1FOS™ vector and the reagents supplied in the CopyControl™ Fosmid Library Production Kit (Epicentre, Cambio Ltd., Cambridge, UK), following the supplier’s recommendations. All clones were picked using a colony picker Genetix QPix2 XT (Genetix Ltd., New Milton, England) and subcultured for 20 h in 384-well plates (Genetix Ltd., New Milton, England) containing Luria Bertani broth (LB) with 12.5 μg/mL chloramphenicol and 20 % glycerol. They were then stored at −80 °C.