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1.9
mismatches). Finally, reads were also aligned to 404 genomes of faecal isolates
including the HMP reference genomes from the gastrointestinal tract (n 372,
downloaded on 19 February 2013 from
66
http://www.hmpdacc.org/HMRGD/) and
the MetaHIT draft sequences (n 32, downloaded on 19 February 2013 from
http://www.sanger.ac.uk/resources/downloads/bacteria/metahit/). After
normalization, a distance score between all pairs of depth proles was calculated as
0.5 (r/2), where r is the Spearman rank correlation between the depth proles.
The symmetrical distance matrix was converted to a co-occurrence cladogram by
using BioNJ
68
(Fig. 3). Clustering with the phages separately did not change their
association to the Bacteroidetes cluster.
Testing for crAssphage in CRISPR spacers. Using pilercr v1.06 (ref. 47), we
screened 2,773 complete prokaryotic genomes from Genbank
35
and 404 faecal
isolates (above), identifying 79,977 and 13,299 CRISPR spacers, respectively. All
spacers were queried by using blastn 2.2.27 with short query parameters
57
against the crAssphage genome. The best global hits from either collection of
genomes (that is along the complete spacer length) are displayed in Fig. 2. Both
these spacers match genomes from the class Bacteriodetes.
Plaque assays. We attempted to isolate crAssphage by isolating Bacteroides-
specic phages from faecal phage lysates using an adapted plaque assay method
68
.
To do this, we obtained 10 phage lysates from faecal samples. A quantity of 1 ml
sodium magnesium (SM) buffer was added to faecal sample and vortexed on high
for 1 h. SM buffer contained (per 500 ml): 2.9 g NaCl (Fisher Scientic, Waltham,
MA), 1 g MgSO
4
7H
2
O (Fisher Scientic) and 25 ml of 1 M TrisHCl pH 7.4
(Sigma-Aldrich, St Louis, MO). Samples were centrifuged for 4 min at 12,000 r.p.m.
(revolutions per minute) (that is, 13.4 relative centrifugal force, r.c.f.) and the
supernatant transferred to a clean tube. A quantity of 50 ml ml
1
of chloroform
(Fisher Scientic) was introduced to the supernatant to arrest growth, vortexed
briey and incubated at 4 C for 20 min. Following cold incubation, samples were
centrifuged for 10 min at 4,000 r.p.m. (that is, 1.5 r.c.f.), supernatant transferred to a
clean tube and 15 ml of 100 units ml
1
DNase (Fisher Scientic) added. Samples
were incubated at 35 C for 1 h and subsequently incubated at 65 C for 15 min. To
separate viral-like particles (VLPs), remaining samples were passed through a
0.45 mm lter (Millipore Corporation, Billerica, MA) attached to a 3 ml syringe
(Becton, Dickinson and Company, Sparks, MD). The resulting lysate was
topped with additional SM buffer plus 16 mM MgSO
4
to reach a volume of 1 ml
and stored at 4 C.
Two gut-associated Bacteroides strains were tested: the annotated host of the
known Bacteroides phages B124-14 (ref. 22) and B40-8 (ref. 26), Bacteroides fragilis;
Table 2 | PCR primer pairs designed to validate the crAssphage genome sequence.
Nr Forward primer sequence Start Reverse primer sequence End
1 5
0
-GTGACGAGAGGTATTGAATGTGGA-3
0
1,785 5
0
-GCTATAAGTCCAGCAGCAAAAGG-3
0
6,793
2 5
0
-TGACTAGCTTGCTTCCATCCT-3
0
6,004 5
0
-GCACTACGTCCATCTTGAGTACCA 11,923
3 5
0
-CAGGTGAACGTAAACCTGTTCCT-3
0
9,512 5
0
-ACTCATACCAGCAAATGAAGGCA-3
0
14,930
4 5
0
-ATGGTGCTCGTGAAATTGCT-3
0
13,526 5
0
-GCTTTACGCTGAGCAATCGT 17,858
5 5
0
-GCACCGGTATTGCAAAGGCT-3
0
17,801 5
0
-CTCCAAATCCTTTGTTTCCACGT-3
0
25,822
6 5
0
-TGCTATTTGGCAAACTGCTGG-3
0
23,445 5
0
-ATCATGCTGACCGTCTTGCT 29,713
7 5
0
-GTAGCGAAGCGGAGCGTTCTA-3
0
28,192 5
0
-TATGGAACGAGCTGCTGGTG 30,897
8 5
0
-ATTCACCAGCAGCTCGTTCC-3
0
30,875 5
0
-TGAATGGCGTTCAGCAGGCT-3
0
36,058
9 5
0
-AGCTATTCCGCCCTCACTCAA-3
0
35,019 5
0
-TGCTAAGATTGGTCGTGTAGCT 40,185
10 5
0
-TGAGGAACTTCTTGCTGACGA-3
0
38,017 5
0
-ACTTAAAGGTGATGCTCGACGT-3
0
43,360
11 5
0
-TCAGGTATTGTTCCATCCTCC-3
0
42,662 5
0
-CAAGATACTAGTTGGAGAGCTGCT 47,956
12 5
0
-CTGCAAAACCAATAGCTGTACCA-3
0
47,220 5
0
-GGTGGTATTGCTCAACCTATTGG 52,314
13 5
0
-AGAGTAGGTTGACCTGGGCCT 50,678 5
0
-AGGTTATGGTGGGCTACAAGAT-3
0
54,585
14 5
0
-TGGTCTTGTGCAGCTTGAGC-3
0
53,477 5
0
-TATGCCCGATGATTGTTGTCCT 58,673
15 5
0
-GACCAGAACGACCTCCACTA-3
0
57,353 5
0
-TCTTGATGGTCGAGTTGATGCT-3
0
62,669
16 5
0
-CACGAATACGTTGTTGCAAACCT-3
0
60,536 5
0
-ATCGGTACTGCACTTGGTGC-3
0
66,345
17 5
0
-ACCAGCCGTAAACATCTTTTCCA-3
0
65,848 5
0
-AGTATTGGAGCAACAGGTGGA-3
0
71,818
18 5
0
-AGCAGGAACAGCTTTACGAGTA-3
0
69,175 5
0
-TTGCTAGTCTTGATGGAGATGGT-3
0
74,902
19 5
0
-GTGGCACTTATTCAGTACCACCA-3
0
74,161 5
0
-CAGAATTAGGCTTCCCATTGAACG-3
0
79,628
20 5
0
-CGAAGTTTAGCAATAGGCTGCCA-3
0
78,705 5
0
-AGGCTCTATTGGTTTGCAGGT-3
0
83,203
21 5
0
-TAGCAAGACGCTCAGCTTCTC 82,364 5
0
-GTTTGCTGAACGTCGTATGTTGAC-3
0
87,950
22 5
0
-TCCATACGTTCTTCAGCTTGATTC-3
0
86,454 5
0
-AGATGATGCTGGTGGAGAAACTT-3
0
91,978
23 5
0
-GTCCAACCTTGCCAAGTAGGA-3
0
91,910 5
0
-TGACCATCAGTACAGATGCGTCTA-3
0
638
24 5
0
-AGCGTCAAGTGCTTCACTTG-3
0
95,395 5
0
-CGAAGTCCACCATCAGCAGT-3
0
3,167
Numbers in the rst column correspond to the bands in Supplementary Table 1. Numbers in the Start and End columns refer to the position on the crAssphage genome.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5498 ARTICLE
NATURE COMMUNICATIONS | 5:4498 | DOI: 10.1038/ncomms5498| www.nature.com/naturecommunications 9
& 2014 Macmillan Publishers Limited. All rights reserved.
and B. thetaiotaomicron (stocks kindly provided by San Diego State University
Microbiology Department, San Diego CA; SDSU-MD accession numbers 939 and
906, respectively).
Phages were isolated using the adapted most probable number method
68
.
2 Bacteorides phage recovery medium was incubated with a 1:1 mixture of host
and phage lysate for 24 h at 37 C. Following incubation, the incubated medium
was subjected to chloroform at 50 ml ml
1
of broth to arrest any living host cells.
Supernatant of the various conditions was spotted atop a fresh lawn of host on
Bacteorides phage recovery medium agar plates. Plates were placed into a GasPak
(BBL) jar and incubated at 37 C for 16 h. Plaques appeared, and 10 pools of 10
plaques each were selected. DNA was extracted and PCR performed using the
primer pairs F4R4, F4R6 and F23R23 as above (Table 2). Although these primer
pairs successfully amplied regions of the crAssphage genome from the faecal
samples F2T1 and TSDC8.2 (Supplementary Table 1), no bands were observed
after application of the same primer pairs to the plaque pools, indicating that these
plaques were probably caused by other Bacteroides-infecting phages.
Phages in metagenomes. To determine the prevalence of the crAssphage genome
across different environments, we downloaded sequencing reads from all 2,944
publicly available shotgun metagenomes available in the MG-RAST database
49
. A
database of 1,193 phage genomes was created by adding crAssphage to all complete
phage genomes from PhAnToMe (http://www.phantome.org). A blastn
57
2.2.27
search of every metagenomic read versus the phage genome database was
performed, and hits were considered if they had at least 95% identity over an
aligned length of 75 bp. Ambiguously aligned reads were discarded.
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Acknowledgements
We thank Alejandro Reyes, Jeffrey Gordon and Forest Rohwer for access to virome
materials and fruitful discussions; Michiyo Wellington-Oguri for initial host predictions;
Anca Segall for help with iVireons; and Cynthia Sears (NIH-R01CA151393) for
screening Bacteroides genomes. B.E.D. was supported by NWO Veni (016.111.075),
CAPES/BRASIL and the Dutch Virgo Consortium (FES0908, NGI 050-060-452); D.R.S.
by was supported BE-Basic (fp0702); and R.A.E. was supported by grants from the
National Science Foundation (DBI-0850356, MCB-1330800, and DEB-1046413). High
performance computation was provided by award CNS-1305112 from the Information
and Intelligent Systems Division of the National Science Foundation to R.A.E.
Author contributions
B.E.D., B.F., J.L.M., R.A.E. performed and analysed the initial cross-assembly; B.E.D.,
D.R.S. assembled crAssphage genome; B.E.D., J.L.M. designed PCR primers; N.C., J.L.M.,
E.A.D. performed long-range PCR experiments; B.E.D., G.G.Z.S., J.L.M. analysed Sanger
sequencing results; B.E.D., J.J.B., V.S., R.K.A., R.A.E. annotated crAssphage genome and
ORFs; R.A.E., phage proteomic tree; B.E.D., bioinformatic host predictions (co-occur-
rence and CRISPRs); S.S., L.B., plaque assays; B.E.D., K.M. analysed public metagenomes;
B.E.D. wrote paper with input from all authors.
Additional information
Accession codes: The crAssphage genome sequence was deposited in the Genbank
Nucleotide sequence database with accession code JQ995537. Sanger sequenced regions
of the PCR products from F2T1 and TSDC8.2 (indicated by the light green regions of the
green bands of Fig. 1) were deposited in the Genbank nucleotide sequence database with
accession codes KM000086 to KM000121 (see Supplementary Table 1).
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing nancial interests: The authors declare no competing nancial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Dutilh, B. E. et al. Unknown sequences in faecal metagenomes
reveal a widely distributed and highly abundant bacteriophage. Nat. Commun. 5:4498
doi: 10.1038/ncomms5498 (2014).
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