Phage G28 was sequenced and assembled by Microsynth AG (Balgach, Switzerland) using Illumina technology (MiSeq (2 × 300 bp), Illumina, Inc., San Diego, CA, USA). Sequencing of E28 was already published [39 (link)]. Shearing of 150 ng gDNA of the mutant E28.G28R3 was performed in a microTUBE AFA Fiber Snap-Cap (Covaris, Inc., Woburn, MA, USA) with the Covaris® S2 DNA shearing technology. To attain a fragment size of 300 bp, shearing was done at an intensity of 5, 10% duty cycle, 200 cycles per burst and a treatment time of 50 s. To prepare next-generation sequencing libraries the NEBNext Ultra™ kit (New England Biolabs Ltd., Hitchin, UK) was used based on the manufacturer’s instructions. A volume of 55.5 µL of sheared DNA was utilized for adapter ligation and USER excision followed by a size selection. Therefore, volumes of Ampure XP (Beckman Coulter GmbH, Krefeld, Germany) beads were adjusted for a 300 bp insert size. NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (New England Biolabs, Ltd., Hitchin, UK) were used for the PCR enrichment and sequencing was carried out on a NextSeq™ system (Illumina, San Diego, CA, USA).
Miseq 2 300 bp
Manufactured by Illumina
Sourced in United States, Germany, Spain
The MiSeq (2 × 300 bp) is a benchtop sequencing system designed for targeted resequencing, amplicon sequencing, and small genome sequencing. It provides up to 15 Gb of output with read lengths of up to 2 × 300 base pairs (bp).
Automatically generated - may contain errors
Lab products found in correlation
Nebnext multiplex oligos for index primers set 1, by Illumina (1 mentions)
Nextseq system, by Illumina (1 mentions)
Microtube afa fiber snap cap, by Covaris (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Axyprep dna gel recovery kit, by Corning (1 mentions)
Hiseq2500 pe250, by Illumina (1 mentions)
Miseq 2x300 bp, by Illumina (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Ultraclean mega prep soil dna kit, by Qiagen (1 mentions)
8 protocols using miseq 2 300 bp
1
Sequencing and Assembly of Phage G28
2
Amplicon Sequencing of Fecal Microbiome
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Fecal samples were prepared for the MiSeq Library, followed by Illumina MiSeq 2 × 300 bp high throughput sequencing and bioinformatics analysis. Briefly, the cDNA library was constructed using a two-step PCR amplification method. Firstly, the target fragment was amplified using specific primers (inner primers) and recovered by glue. Then, the recovered product was used as the template for secondary PCR amplification (outer primers). The inner primer was F-(5′-TTC CCT ACA CGA CGC TCT TCC GAT CT-specific primer-3′) and R-(5′-GAG TTC CTT GGC ACC CGA GAA TTC CA- specific primer -3′), and the outer primers was F-(5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC AC- barcode – TCT TTC CCT ACA CGA CGC TC -3′) and R-(5′-CAA GCA GAA GAC GGC ATA CGA GAT- barcode – GTG ACT GGA GTT CCT TGG CAC CCG AGA-3′). All PCR products were recovered using AxyPrepDNA gel recovery kit (AXYGEN, Hangzhou, China), and quantitative analysis was conducted using FTC-3000 real-time PCR. The samples were mixed according to the molar ratio, and the library was prepared for standard Illumina sequencing using HiSeq2500 PE250 (Illumina, United States).
3
Hybrid Genome Assembly and Annotation
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WGS was performed on SP strain C5347 using PacBio Sequel and Illumina Miseq 2 × 300 bp platforms prior phenol-chloroform DNA extraction, as previously described [44 (link)]. Raw PacBio reads were assembled using Canu [45 (link)] with default parameters and setting an estimated genome size of 3 Mb. The resulting assembled contigs were then polished as follows: First, Illumina raw reads were quality-trimmed using Trimmomatic [46 (link)] and aligned against the assembled PacBio contigs using Bowtie2 [47 (link)]. Then, the resulting bam files were used to fix individual base errors, indels and local missassemblies using Pilon [48 (link)].
Resulting genes on the assembled contigs were predicted using Prodigal [49 (link)]. tRNA and rRNA genes were predicted using tRNAscan-SE [50 (link)], ssu-align [51 ] and meta-rna [52 (link)]. Predicted protein sequences were compared against the NCBI nr database using DIAMOND [53 (link)], and against COG [54 (link)] and TIGFRAM [55 (link)] using HMMscan [56 (link)] for taxonomic and functional annotation.
Resulting genes on the assembled contigs were predicted using Prodigal [49 (link)]. tRNA and rRNA genes were predicted using tRNAscan-SE [50 (link)], ssu-align [51 ] and meta-rna [52 (link)]. Predicted protein sequences were compared against the NCBI nr database using DIAMOND [53 (link)], and against COG [54 (link)] and TIGFRAM [55 (link)] using HMMscan [56 (link)] for taxonomic and functional annotation.
4
Viral Pathogen Enrichment and Sequencing
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Before library preparation, NA were retrotranscribed to cDNA, tagged and complemented to obtain dsDNA. This viral randomly tagged dsDNA was then amplified (25 cycles) to obtain the sufficient amount of DNA for the preparation of libraries, as previously described (Fernandez-Cassi et al., 2018 (link)). Libraries were prepared in duplicate using KAPA HyperPrep Kit following the instructions provided by the manufacturer (Roche-Kapa Biosystems). One replicate of the libraries was hybridized with probes designed to capture sequences from vertebrate viral pathogens (VirCapSeq Enrichment Kit, Roche). Two negative controls were also processed, one of them with the enrichment kit.
After the capture, quality and concentration were re-checked and sequencing of the libraries from the captured, non-captured, and negative controls was performed (Illumina Miseq 2x300bp).
Clinical samples were sequenced in an independent Illumina Miseq 2×300bp run (in this case, consensus sequences were assembled with reads produced from another Illumina TruSeq high coverage sequencing run on same samples, and are already available at GISAID as EPI_ISL_418860 and EPI_ISL_418861, respectively).
After the capture, quality and concentration were re-checked and sequencing of the libraries from the captured, non-captured, and negative controls was performed (Illumina Miseq 2x300bp).
Clinical samples were sequenced in an independent Illumina Miseq 2×300bp run (in this case, consensus sequences were assembled with reads produced from another Illumina TruSeq high coverage sequencing run on same samples, and are already available at GISAID as EPI_ISL_418860 and EPI_ISL_418861, respectively).
5
Microbial Community Analysis of Enriched Sediments
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To analyse the microbial community of the enriched sediments, the V3–V4 region of the 16S rRNA gene was amplified using primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785 R (5′-GACTACHVGGGTATCTAATCC-3′) with Illumina adapter sequences as overhang [48 (link)]. PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs), with the following cycling parameters: initial denaturation at 98 °C for 1 min, followed by 27 cycles of denaturation at 98 °C for 30 s, annealing at 57 °C for 30 s and an extension at 72 °C for 30 s. The final extension was performed for 5 min at 72 °C. Library preparation and sequencing (Illumina MiSeq 2×300 bp) were performed by Eurofins Genomics, Konstanz, Germany.
Amplicon sequence variants (ASVs) were analysed using the dada2 pipeline in R [49 (link)]. Reads were filtered and merged using the default parameters of the pipeline, except the following parameters during the merging steps: trimLeft=c(17,21), truncLen=c(272,216) and truncQ=4. Singletons and chimaeras were removed. Taxonomy was assigned using the Silva small subunit rRNA database v138.1 [50 (link)]. Alpha diversity indices were calculated using the phyloseq package for R [51 (link)].
Amplicon sequence variants (ASVs) were analysed using the dada2 pipeline in R [49 (link)]. Reads were filtered and merged using the default parameters of the pipeline, except the following parameters during the merging steps: trimLeft=c(17,21), truncLen=c(272,216) and truncQ=4. Singletons and chimaeras were removed. Taxonomy was assigned using the Silva small subunit rRNA database v138.1 [50 (link)]. Alpha diversity indices were calculated using the phyloseq package for R [51 (link)].
6
Metagenomic analysis of insect gut and fat
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Hindgut and fat body were ground with a sterile plastic pestle. Hindgut total DNA was obtained using the JetFlex Genomic DNA Extraction kit (Genomed, Germany) following the manufacturer’s recommendations, adding lysozyme (20 mg/ml) to the cell lysis buffer to break Gram-positive bacterial cell wall, and used for metagenomic sequencing using the Illumina MiSeq (2 × 300 bp) technology at the FISABIO (Valencia, Spain). Total DNA from the fat body was extracted following the protocol described in Llop et al. (1999) (link) with an additional phenolization and was used for qPCR analyses. DNA was quantified with Qubit (Thermo Fisher, Massachusetts, United States).
7
Geothermal Sediment Microbiome Analysis
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The green sediment sample was collected around a steam vent at the geothermal field of Los Azufres, Mexico, in April 2013 (Figure 1 ) (19.78170609819753 N, −100.65805210414699 W). The sediment sample (Figure 1 ) had a temperature of 67 °C and a pH of 3. DNA was isolated using the UltraClean Mega (Prep) Soil DNA Kit (MoBio Laboratories, Inc., Carlsbad, CA, USA). The sequencing was performed on an Illumina Miseq 2 × 300 bp at Macrogen, Inc., Seul, Republic of Korea.
8
High-throughput antibody sequence analysis
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