title: Mars Oasis Soil Metagenome
design: The four soil samples (75 g each) were each suspended in 1% SDS solution (25 ml), to which 0.05 g of   glucanex and glucanase had been added. The suspensions were vortexed for a few seconds and then incubated at 37 °C for 4 h. They were cooled and filtered (1 mm) and 250 µg of RNAase was added to each  solution.  The  solutions  were  centrifuged  six  times  at  7,500  rpm  for  15  min.  After  each  centrifuge  run,  the  supernatant  (12  ml)  was  decanted.  3  M  sodium  acetate  at  pH  7.0  (1.2  ml)  and    ethanol (26.4 ml) was added to each aliquot of the supernatant, which was centrifuged at 10,000 g for  10  min.  The  pellet  of  DNA  was  drained  and  dried  for  several  minutes.  TE  buffer  (0.1  ml)  was added to each pellet, which was then incubated at 4 °C for 16 h. All of the TE buffer solutions were   combined (~ 240 µl), mixed with an equal volume of loading buffer, and ran out in a large-welled 1%   low melting point agarose gel (Sigma-Aldrich) at 20V overnight. The gel ran for 48 h, after which a block of agarose containing the target DNA, which had advanced 14-17 mm, was excised from the gel with a sterile scalpel. A size standard was used to select the region of the gel containing 35-45   kbp fragments. The agarose containing the target nucleic acids was then kept at 4 °C for 60 h.    The agarose was digested in Gelase according to the manufacturers’ instructions and enzymes were then denatured by heating to 60  °C for 30 seconds. DNA was precipitated in three separate aliquots and was re-suspended in TE buffer (4.5 µl). The concentration of DNA (3 x 20 ng µl-1  aliquots) was determined by running against known standards in 1.5% agarose gels (1 h at 120 V). The DNA was then end-repaired by combining aliquots (12 µl) on ice with 10 × end-repair buffer (1.85 µl), 2.5 mM   dNTP  mix  (1.85  µl),  10  mM  ATP  (1.85  µl)  and  end-repair  enzyme  mix  (0.92  µl).  The  mixture  was incubated at room temperature for 45 min and then at 70 °C for 10 min. The DNA, consisting of a solution  containing  240  ng  of  DNA,  was  then  ligated  into  the  pEpiFOS-5  fosmid  vector  (EpiCentre,  Madison, WI) by combining it with sterile water (2 µl), 10 × fast link ligation buffer (3 µl), 10 mM ATP  solution (3 µl), fastlink ligase (3 µl) and vector (1 µl). The ligation reaction was then incubated at 4 °C for 7 d.  The ligation mix was heated to 70 °C for 10 min and the fosmid clones were packaged into lambda phages  using  MaxPlax  lambda  packaging  extracts  according  to  the  manufacturer’s  instructions   (Epicentre,  Madison,  WI).  This  process  yielded  three  aliquots  (1.025  ml)  of  cloned  cells.  The packaged library was transduced into E. coli EPI-100, and E. coli transformants were selected on LB agar supplemented with 12.5 µg ml-1 chloramphenicol. After determining the number of viable cells present,  aliquots  (200  µl)  were  spread  onto  dry  Luria  broth  (100  ml)  containing  12.5  µg  ml-1 chloramphenicol in 47 Petri dishes (150 mm diameter). Chloroformed phage (0.5 ml) was added to EPIFOS cells (5 ml, OD 1.0) and were incubated at 37 °C for 20 min. Aliquots (100 µl) were added to each plate, spread, and grown at 37 °C for 17 h. Colonies were picked into individual wells of 96 well plates containing Luria broth with 12.5 µg ml-1 chloramphenicol (40 µl). The plates were incubated at 37 °C for 17 h before sterile glycerol (10% v/v) was added to each well and the plates covered with plastic  seals  prior  to  storage  at  -80  °C.  All  procedures  described  above  took  place  under  a  sterile hood.    Quality control was established by end sequencing 20 random fosmids using pEpiFOSTM-5 forward  and reverse end sequencing primers to ensure environmental DNA had been successfully    incorporated, from microorganisms that one might expect to find in this extreme environment . One   full fosmid was also sequenced. Primers were used to identify specific sequences from the fosmid   library. The metagenomic library was screened using a range of primers for viral (Cyanophage   CPS4GC, CPS5 Fuller et al., 1998; Wilson et al., 1999 and Phycodnaviridae AVS1, AVS2 Chen and   Suttle, 1995), fungal (ITS1F / ITS4F; White et al. 1990; Gardes and Bruns 1993), phosphonate (Gilbert   et al. 2009) and N cycling (nosZ-F / nosZ-R, nirS1F / nirS6R and nifHF / nifHRb; Thröback et al. 2004,  Rösche & Bothe 2005) genes. A selection of E. coli cells containing fosmids were screened for antibiotic production.   Cells from 25 plates (10% of the total) were combined (to favour depth of sequencing rather than coverage)  and  cultured  in  Luria  broth  with  12.5  µg  ml-1  chloramphenicol  overnight  in  a  shaking incubator at 37  °C  until an OD of 0.8 was obtained. The cells were centrifuge-concentrated and used to  construct  a  10,000  Gbp  metagenomic  library  for  454  pyrosequencing.  Fosmids  were  extracted from  E.  coli  cells  using  the  QIAGEN  Plasmid  Midi  Kit  (QIAGEN  Plasmid  Midi  Kit,  Cat.  No.  12145. QIAGEN)  and  then  treated  with  ATP-dependent  Exo-nuclease  (Plasmid-SafeTM  ATP-Dependent Dnase, 10U/ul 10,000 U, Cat. No. E3110K, Epicentre). Extracts were sequenced by Macrogen (South Korea)  according  to  the  emPCR  Method  Maual  –  Lib-L  MV  (Anon,  2009a)  and  the  Sequencing Method Manual (Anon, 2009b).