454 gs flx system

In 2014, a total of ~13,000 unfed ticks were collected from the wild in a field in Guertu County, Wusu City, located in the Western Tianshan Mountainous areas in Xinjiang Province, China (Supplementary data). The ticks were classified as D. nuttalli and grouped into 76 pools of 100–200 individuals each according to the sampling locations. Eight pools of tick samples were randomly chosen and used for library preparation, which were sequenced on a Roche 454 GS FLX system (GS-FLX, Roche Applied Science) for metagenomic sequencing (Supplementary data). The complete genomic sequences of GTV were obtained by gap-filling RT-PCR with specific primers designed based on corresponding contig sequences. The terminal sequences were confirmed by rapid-amplification of cDNA ends (RACE)^{54 (link)}.

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.

The bacterial genomic DNA was amplified with 343F (5’- TACGGRAGGCAGCAG-3’) and 798R (5’- AGGGTATCTAATCCT -3’) primers specific for the V3-V4 hypervariable regions of the 16S rRNA gene [15 (link)]. Barcodes that allowed sample multiplexing during pyrosequencing were incorporated between the 454 FLX Titanium adapter and the 5’ end of the forward primer. The thermocycling steps were as follows: 95°C for 5 min, followed by 20 cycles at 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and a final extension step at 72°C for 10 min.
Each PCR reaction was performed in a 50 μl system, and the products were extracted with the QIAquick gel extraction kit (Qiagen) and quantified on a NanoDrop 2000C spectrophotometer and Qubit^® 2.0 Fluorometer (Life technologies).
The samples were pooled in an equal amount and sequenced on a Roche 454 GS FLX+ System (Roche, Basel, Switzerland) according to the manufacturer’s recommendations. The sequencing was started from the the 454 FLX Titanium adapter.

Purified DNA extracts from rhizosphere soil samples were initially submitted to Macrogen, Inc. (Seoul, South Korea) for 454-pyrosequencing (Roche) using 16S rRNA as a gene target. The selection of primer set and sequencing of 16S rRNA gene libraries were according to the Macrogen, Inc. protocol and recommended for Roche 454 GS-FLX System using Titanium Chemistry (454 Life Sciences). Briefly, 16S rRNA gene libraries were prepared by PCR using the universal bacterial primer UNI_AMP-27F (5′-Zxxx GAG TTT GAT CMT GGC TCA G-3′ and UNI_AMP-518R (5′-K WTT ACC GCG GCT GCT GG-3′) (Lee et al., 2010 (link)), where Z and K represent two pyrosequencing primers (CCATCT CAT CCC TGC GTG TCT CCG ACT CAG and CCTATC CCC TGTG TGC CTT GGC AGT CTC AG), and xxx was designed for the sample identification barcoding key. The PCR reaction was as follow: a hot start at 95°C for 3 min, PCR amplification was carried out for 35 cycles at 94°C for 15 s, 55°C for 45 s, and 72°C for 1 min. A final extension step was carried out at 72°C for 8 min. The 16S rRNA gene libraries were sequenced by with Roche 454 GS-FLX System using Titanium Chemistry (Roche Diagnostics Corporation, Life Sciences, Branford, CT, United States).

The genome was sequenced with the Roche 454 GS FLX system by using shotgun strategy. The reads were assembled with Roche GS De Novo assembler software. Contigs assembly was assisted by previously generated restriction maps [13] (link). A few regions that were not assembled into the contigs were further amplified by PCR, cloned and sequenced. The genome sequence data was uploaded to GenBank (GenBank accession number: KF611977).
Hypothetical ORFs were predicted by softberry FGENESV program (http://www.softberry.com/berry.phtml) [58] (link) to contain the standard ATG start, and a stop codon and potentially encode at least 50 amino acids. Gene-parity plot analysis [13] (link) was performed using Microsoft Office Excel to draw scatter diagram with using BusuNPV ORFs number as the X-axis and other baculovirus ORFs as the Y-axis. Gene annotation and comparisons were done with NCBI protein-protein BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Repeat structures were detected by BLAST alignment of two sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The identity among homologous genes was done with MegAlign software using clustalW with default parameters. Regulatory regions and promoter motifs were identified as described previously [29] .
Restriction sites were predicted by MapDraw software. Genome map framework drawn with genomeVX [59] (link).

The resulting 16S V1–V3 amplicon libraries were sequenced by using a Roche 454 GS FLX+ system and reagents (Roche 454 Life Sciences Corporation, Branford, CT, USA). Genomic DNA from Microbial Mock Community A, Even, Low Concentration (HM-278D v3.1, BEI Resources) obtained as part of the Human Microbiome Project [21 (link)], was diluted 10 times and used as a positive control for PCR and pyrosequencing of 16S amplicons.
The pyrosequencing data analysis pipeline was based on the Quantitative Insights Into Microbial Ecology (QIIME) pipeline version 1.8 [15 (link)]. The 454 read data were subjected to quality processing, chimera filtering, and removal of singleton reads. The taxonomic classification of the quality-processed reads was based on the closed reference clustering of sequences into operational taxonomical units (OTUs), using the UCLUST tool [22 (link)] with a sequence identity level of 97%. The read clusters were further assigned to taxonomies by using the Ribosomal Database Project (RDP) classifier [17 (link)] with a confidence level of 80%. The bacterial community diversity within a sample (α-diversity) was assessed by using the R-package vegan (https://cran.r-project.org/web/packages/vegan). The diversity between samples (β-diversity) was analyzed by using the QIIME implementation of UniFrac [19 (link)] and the weighted UniFrac distance as a measure of β-diversity.