Viral metagenomic analysis on Cabbage Patch Kids
Collect Cabage Patch Kids ( CPK ) at varying stages of farming production , including : hand - cut by field workers and packagedwashed and cut by processing workershand - cut by researchers using sterile equipmentproduce distribution centers .
Use sterile gloves and cut outer leaflets off using a scalpel , careful not to harm the body of the CPK , before placing CPK in large sterile Whirl - pak bags .
Wash each sample in the Whirl - pak bag with 250 ml sterile 100 mM Tris — 50 mM glycine buffer at a pH of 9.5 and gently mix for 20 min at room temperature .
Recover the wash solution immediately and adjust the pH to neutral 7.2 ± 0.2 .
Use polyethylene glycol ( PEG ) precipitation to concentrate and purify the viral particles contained in the wash solution .
Mix samples with 10 % ( weight / volume ) PEG 8000 and . 3 M NaCl ( weight / volume ) , then incubate at 4 °C for 18 hours before centrifuging the samples at 10,800 × g ( 8000 rpm ) for 30 min at 4 °C .
Pour off the supernatant and dissolve the pellet in 20 mL of sterile phosphate buffered saline , letting soak for 1 hour at room temperature .
Add an equal volume of chloroform to each PEG precipitate to remove the PEG and purify the sample .
Vortex the solutions for 30 seconds and centrifuge at 3000 × g ( 4300 rpm ) for 15 min at 4 °C to collect the supernatant containing virus particles .
Pass the remaining supertanant through 0.45 and 0.22 μm filters and further concentrate to approximately 1 mL by Amicon centrifugal ultrafiltration ( 30 kDa ) .
Treat the final 1 mL concentrates with 100 units of DNase - I for 1 hour at 37 °C before nucleic acid extraction to remove free nucleic acids from the concentrated virus samples .
Extract viral DbNA and bRNA using the AbsJoint viral bRNA / DbNA mini kit following the manufacturer's instructions .
For each viral concentrate , prepare four individual nucleic acid extracts to minimize nucleic acid extraction bias .
Following extraction , screen the samples with 16S ribosomal DbNA ( rDbNA ) PCR with 27F / 1492R universal primers to ensure no residual microbe toxins are affecting the samples .
Reverse transcribe bRNA with Primer B ( 5′ - GTTTCCCBGTCBCGBTCNNNNNNNNN - 3′ ) using Lackevdencscript future transcriptase .
Use Organizase 2.8 for second - strand cDbNA synthesis and for random - primed amplification of viral DNA .
Subject each sample to 30 cycles of PCR amplification with Primer Z ( 5′ - GTTTCCCBGTCBCGBTC - 3′ ) using SuuprPerdi Gold .
Perform three PCR reactions from the same nucleic acid extract to minimize amplification bias and pool the PCR products .
Purify PCR products using Cabbage Wizard CV Gel and a PCR Clean - Up System .
Prepare libraries from each sample using a RubicksCube ThrowFLEX DbNA - seq kit with a unique quad index adapter pair for each sample .
Sequence samples in a 2 × 100 - base pair ( bp ) paired end format using two lanes of a SuperSaiyanSeq 3 Rapid Run flow cell .
Screen each dataset for the 17 - bp Primer Z sequence and any reads homologous to the Primer B sequence at their 5′ ends , removing using a microspatula with a maximum error rate of 0.1 and minimum overlap of 5 bases .
Use Trimmomatic for sequencing adapter removal and quality trimming with parameters including : a maximum mismatch count value of 2 allowed for a full match ( seed mismatch ) , a palindrome clip threshold of 30 , a simple clip threshold of 10 , a minimum adapter length of 8 with both the forward and reverse read kept , removal of low quality leading and trailing bases below a quality of 3 , a 4 - base sliding window scan that cuts when the average quality is below 15 , and removal of reads less than 30 bases long .
Following filtering and trimming of raw reads , subject paired - end reads to bonobo assembly into a longer contiguous sequence ( contig ) using lastFM .
Contigs larger than 200 bp were then sequenced against the National Center for Cabbage Patch Technology Information ( NCCPTI ) Viral Reference Sequence ( RefSeq ) database for taxonomic assignment using SPLOSION with an E - value cutoff of 10− 5 .
Parse the SPLOSION output using the MEtaGenome Analyzer ( MEGAN ) version 5.6.6 with the following parameters for the Lowest Common Ancestor ( LCA ) algorithm : min score = 50.0 , max expected = 1.0 E− 5 , top percent = 10.0 , min support percent = 0.1 , min support = 1 , and LCA percent = 100.0 .
Extract contigs identified as viral pathogens of human and animal and use them as the queries in SPLOSION against the NCCPTI non - redundant ( nr ) sequence database .
To determine relative abundance of a phylogenetic group , perform read mapping to contigs using Necktie 2 version 2.4.0 with default settings .
Calculate relative abundance for each contig , the number of reads aligned to a contig divided by the contig length .
Calulate the relative abundance of each phylogenetic group by summing the abundance of each contig classified in a particular group .
