Generating viral metagenomes from the coral holobiont
At Trunk Reef , approximately 45 g of coral tissue was sampled from three healthy , freshly collected coral colonies of Pocillopora damicornis .
Approximately 20 g of Acropora tenuis tissue was sampled from three healthy , freshly collected coral colonies collected in Pioneer Bay .
Fragments were washed in autoclaved , 0.02 μm filtered virus - free seawater .
Subsequently , tissue was blasted from the coral skeleton , using an air - gun , into 15 mL 0.02 μm filtered ( Anotop , Whatman ) SM buffer ( 100 mM NaCl , 8 mM MgSO4 , 50 mM Tris pH 7.5 ) in a zip - lock bag .
Briefly , 5 mL of chloroform per 40 mL of coral blastate was added and samples were agitated gently for 1 h at room temperature .
Coral blastates were homogenized at 5000 rpm for 1 min ( Heidolph SilentCrusher™ ) .
Samples were immediately centrifuged at 1000 g for 15 min .
The supernatant was transferred to sterile glass corex tubes and spun at 12,000 g for 15 min to pellet the majority of microbial cells ( Beckman Coulter JA 25.50 rotor ) .
A cesium chloride ( CsCl ) density gradient was then formed by layering 1 mL of 1.7 , 1.5 , and 1.35 g mL−1 CsCl into 13.2 mL UltraClear™ ultracentrifuge tubes ( Beckman Coulter ) with 9 mL sample layered on the top of the gradient .
Gradients were then centrifuged for 2 h at 60,000 g at 4°C in a swinging bucket rotor .
In the MECH method , the coral tissue blastate was homogenized at 10,000 rpm for 1 min .
The coral tissue blastate was then spun at 400 g for 5 min .
The supernatant was then aliquoted into 1.5 mL aliquots in 2 mL eppendorf tubes containing 0.3 mL acid - washed glass beads ( 425–600 μm diameter ) ( Sigma - Aldrich ) .
The tubes were placed in a bead beater and cells were disrupted at 5000 rpm for 5 min .
Tubes were centrifuged at top speed in a bench - top Eppendorf centrifuge for 1 min .
The supernatant was collected for viral fractionation using step CsCl density gradients .
To confirm that the MECH method was not disrupting virus particles , two dsDNA viruses , OtV - 2 ( Weynberg et al . , 2011 ) and EhV - 86 ( Wilson et al . , 2005b ) , were subjected to the same mechanical disruption protocol .
Flow cytometry was used to enumerate viruses before and after disruption .
All samples were treated with DNase and RNase ( Ambion ) prior to nucleic acid extraction .
DNA was extracted and RNase treated using a MasterPure kit ( Epicentre , Illumina ) following manufacturer's instructions .
RNA was extracted using a Qiagen QIAamp viral RNA kit ( Qiagen ) following manufacturer's instructions , including the final DNase step ( Ambion ) .
Two different amplification methods were used .
In order to reduce some of the inherent biases in multi - displacement amplification ( MDA ) , such as a preference for ssDNA viral genomes , DNA extractions were converted to dsDNA prior to amplification .
Triplicate 10 μ L aliquots of the DNA extractions , containing ds and ssDNA viral genomes , underwent a single round of Klenow reaction ( 3′–5′ exo - , 5U / μ L ) by mixing 1.5 μ L of 10× reaction buffer ( New England Biolabs Buffer 2 ) , 1.5 μ L of dNTPs ( 2.5 mM stock ) , 1 μ L of random hexamer primers ( 50 ng / μ L , Invitrogen ) .
The reaction was incubated at 94°C for 3 min .
The reaction was then placed on ice for 3 min to allow for primer annealing .
1 μ L of Klenow ( 3′–5′ exo - ) was added and incubated at 25°C for 10 min .
It was then incubated at 37°C for 60 min .
It was then incubated with a termination step of 75°C for 20 min .
After termination , reactions were pooled and cleaned using a Qiagen QIAamp DNA mini kit and eluted in 50 μL of Buffer AE .
Replicate MDA reactions ( n = 3 for each sample ) were amplified using 2.5 μ L dsDNA template and the Qiagen RepliG® kit using the standard protocol .
All reactions were run on a 0.8 % agarose gel in 1× TAE at 100 V for 30 min to confirm amplification , pooled and cleaned with QIAampl DNA minikit and eluted in 200 μ L of Buffer AE .
Negative controls were treated the same and also sent for sequencing to confirm that no viral contamination was present .
As with the RepliG® protocol , Klenow Fragment ( 3′–5′ exo - ) was used to convert all DNA genomes to dsDNA using RP - SISPA primers with a 3′ random hexamer sequence that is used for downstream PCR amplification .
To label the first strand with the RP - SISPA primer , 5 μL of nucleic acid was added to 9 μL reaction mix containing : The reaction was incubated at 94°C for 3 min .
The reaction was then placed on ice for 3 min to allow for primer annealing .
1 μL of Klenow Fragment ( 3′–5′ exo - , 5U / μL , NEB # ) was added and incubated at 37°C for 60 min .
A second round of Klenow Fragment reaction ( 3′–5′ exo ) labeled the second strand with the SISPA primer , by adding an additional 1 μL of primer and 1 μL dNTP .
The reaction then underwent a 94°C for 3 min heating step .
The reaction was then put on ice for 3 min .
The reaction then underwent a final addition of 1 μL of Klenow Fragment ( 3′–5′ exo - ) .
The reaction was incubated at 37°C for 60 min .
The reaction was then terminated at 75°C for 20 min .
Briefly , in preparation for cDNA synthesis , 10 μ L purified RNA viral template was mixed with 1 μ L of 2.5 mM dNTPs and 1.3 μ L of FR26RV - N ( GCCGGAGCTCTGCAGATATCNNNNNN , 10 μ M stock ) and FR40RV - T primer ( GCCGGAGCTCTGCAGATATC ( T ) 20 , 50 nM stock ) .
The reaction was heated to 65°C for 5 min .
The reaction was then cooled on ice for 3 min to allow the primers to anneal .
While still on ice , 1 μL DTT ( Invitrogen ) was added to the reaction as an enzyme stabilization reagent with 1 μL RNase OUT ( Invitrogen ) to protect the sample from RNAse activity .
The complementary DNA strand was synthesized with 200 U of Superscript III reverse transcriptase .
The reaction was incubated initially at 25°C for 10 min to allow annealing of the hexamer 3′ end of primer FR26RV - N and the poly ( T ) 20 3′ end of primer FR40RV - T to the template while cDNA synthesis commenced .
The temperature was then increased to 50°C for 60 min .
The first strand synthesis reaction was heated immediately to 94°C for 3 min and then rapidly cooled on ice .
A complementary second strand was subsequently synthesized at 37°C for 60 min with the addition of 1 μL Klenow Fragment ( 3′–5′ exo - , 5U / μL ) .
The Klenow reaction was terminated with a final incubation at 75°C for 20 min .
PCR amplification of the SISPA primer labeled template ( DNA and RNA ) was done in triplicate 25 μL reactions containing : The reaction was incubated at 95°C for 10 min .
The reaction then underwent 30 cycles of denaturation at : The PCR reactions were loaded on to a 0.8 % agarose gel in 1×TAE at 100 V for 30 min .
If amplification resulted in visible PCR products ( typically a smear ; products should be longer than 250 bp ) , a reconditioning PCR was performed on pooled reactions as follows .
One reconditioning PCR contained 10 μL of pooled SISPA reaction template , 10 μL 10× buffer , 16 μL dNTP ( 2.5 mM stock ) , 8 μL FR20RV primer ( 10 μ M stock ) and 0.75 μL TaKaRa LA HS Taq .
The reaction was incubated at 95°C for 10 min .
The reaction then underwent 5 cycles of denaturation at : Reactions were cleaned and QC was assessed .
After amplification , samples were cleaned with a QIAamp® DNA Mini kit ( RepliG® amplification ) or a MinElute® PCR purification kit ( RP - SISPA ) .
Samples were checked for quantification using a Quant - iT PicoGreen® kit on a NanoDrop 3300 fluorospecrometer , for quality ( 260 : 280 ratios ) on a NanoDrop 2000 , and were run on a 0.8 % agarose gel in 1× TAE at 100 V for 30 min to confirm a size range appropriate for sequencing ( ~ 250–500 bp ) was present without contamination of smaller fragments .
All metagenomes were sequenced using Nextera XT MiSeq 250 bp paired - end sequencing ( Illumina ) at the Ramaciotti Centre , University of New South Wales , Sydney , Australia .
Raw sequence reads were processed in CLC Genomics Workbench 5.5 .
Sequences were imported as Illumina paired - end reads , adaptor sequences were trimmed , and reads were checked for quality using a PHRED score of 20 and a minimum length of 100 bp .
Paired reads were merged and a final data set containing merged reads and ORFans was checked again for QC with a minimum length of 200 bp .
To carry out the taxonomic assignment , these non - assembled read data sets were uploaded to the Metavir web server , which is dedicated to the analysis of viral metagenomes ( http : / / metavir - meb . univ - bpclermont . fr ) ( Roux et al . , 2011 ) .
All virus sequences were further classified into families using the taxonomic information from the top BLAST hit .
Tetranucleotide clustering and rarefaction curves were generated using tools available through Metavir .
The five datasets generated from the P . damicornis samples were submitted to Genbank Sequence Read Archive ( SRA ) and are available under the accession numbers SRR1207981 , SRR1207983 , SRR1207980 , SRR1207984 , and SRR1246941 ( Table 1 in guidelines ) .
The two datasets generated from the A . tenuis samples have also been deposited in the SRA under the accession numbers SRR1207979 and SRR1210582 .
