First, viral RNA was isolated from infected neurospheres using iPrep PureLink Virus Kit (Thermo Fisher Scientific) and the cDNA synthesis reaction was performed using cDNA Synthesis System Kit (Roche), with random primers. After, PCR was done using specific primers to Asian ZIKV genotype sequence to get amplicons for sequencing. Amplicons were fragmented by enzymatic digestion and libraries built by the automated AB Library Builder System (Applied Biosystems). Libraries were submitted to emulsion PCR using the automated Ion OneTouch 2 platform. The emulsion PCR reaction was loaded on a 318v2 chip and the sequencing reaction was performed on the Ion PGM™ (Thermo Fisher Scientific, USA).
Ab library builder system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AB Library Builder System is a laboratory instrument designed for the automated construction of DNA libraries for next-generation sequencing applications. The system performs essential steps in the library preparation process, including fragmentation, end repair, adapter ligation, and size selection, to generate high-quality DNA libraries from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer high sensitivity chip, by Agilent Technologies (3 mentions)
Ampure xp reagent, by Beckman Coulter (2 mentions)
Ion pi hi q chef kit, by Thermo Fisher Scientific (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Ez bead system, by Thermo Fisher Scientific (2 mentions)
Solid 5 500w system, by Thermo Fisher Scientific (2 mentions)
Cdna synthesis system kit, by Roche (1 mentions)
Iprep pure link virus kit, by Thermo Fisher Scientific (1 mentions)
Ion pgm, by Thermo Fisher Scientific (1 mentions)
Platinum pcr supermix high fidelity, by Thermo Fisher Scientific (1 mentions)
Ion library taqman quantitation kit, by Thermo Fisher Scientific (1 mentions)
Ion torrent next generation sequencing platform, by Thermo Fisher Scientific (1 mentions)
Ion chef system, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Clc genomics workbench software, by Qiagen (1 mentions)
Agencourt ampure xp reagent, by Beckman Coulter (1 mentions)
Ion pi hi q sequencing 200 kit, by Thermo Fisher Scientific (1 mentions)
Ion pi chip v3, by Thermo Fisher Scientific (1 mentions)
16 protocols using ab library builder system
1
ZIKV Genome Sequencing from Infected Neurospheres
2
Exome Capture Library Preparation
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Genomic DNA was prepared for exome capture according to the protocol included with the Ion Xpress Plus Fragment Library Kit for the AB Library Builder (Life Technologies, Carlsbad, CA) using the AB Library Builder System (Applied Biosystems, Foster City, CA). The DNA solvent was exchanged with pure water by ethanol precipitation. Enzymatic shearing was performed using 1–2 μg genomic DNA per sample. Sheared DNA was purified using the Agencourt AMPure XP Reagent (Agencourt, Boston, MA) with a target size peak of 350 bp, followed by adaptor ligation (A1 and P1). The adaptor-ligated genomic DNA fragments were then eluted in 45 μL of low TE buffer and amplified by PCR using 200 μL of Platinum PCR Supermix High Fidelity (Life Technologies), 5 μL of 50 μM library amplification primer mix, and 45 μL ligated DNA. The thermal cycling protocol was initial denaturation at 95°C for 5 min, followed by 7–8 cycles at 95°C for 15 s, 58°C for 15 s, and 72°C for 60 s. The amplified library was purified and eluted in 50 μL of low TE buffer using AMPure XP Reagent.
3
Viral Genome Sequencing using Ion Torrent PGM
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Viral genomes were recovered using the Ion Torrent PGM [11 (link)]. Briefly, the cDNA synthesis reaction was performed using an in-house protocol with random primers. Samples were fragmented by enzymatic digestion and libraries built by the automated AB Library Builder System (Applied Biosystems). To normalize the number of molecules required for emulsion PCR, a quantitation step was performed using the automated Ion OneTouch 2 platform. The emulsion PCR reaction result comprising barcoded-pooled samples was loaded on a 318v2 chip and the sequencing reaction was performed on the PGM device. Raw reads were assembled using Mira v4.0 [12 (link)] (Additional file 1 : Text S1). The six CHIKV genomes generated here have GenBank accession numbers: KP164567–KP164572.
4
Genomic Surveillance of Influenza A
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Lineage typing and sequence analysis were performed by laboratory staff blinded for epidemiological data. RT-PCR products was used in library preparation performed by AB Library Builder system (Applied Biosystems). Each genome library of about 300-bp fragments was quantified with the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific) and template preparation was performed by the Ion Chef system (Thermo Fisher Scientific).
Sequencing was performed using the Ion Torrent next generation sequencing platform with the reference sequence for H3N2 accessed from GenBank. Bioinformatic analysis was performed with the web-based platform INSaFlu and consensus sequences of each InfA genome were obtained [113] . For comparison, samples obtained at primary healthcare centres in the same region, during the same season, were also included. A phylogenetic tree was constructed using the maximum likelihood method in Mega® Version 7. Bootstrap values were obtained from 500 replicates and displayed on nodes if >70%. The modelling process consisted of the following consecutive steps:
(1) Identifying key variables with a potential influence on in-hospital transmission of influenza.
(2) Construction and technical validation of the model.
(3) Selecting the model scenarios of interest.
Sequencing was performed using the Ion Torrent next generation sequencing platform with the reference sequence for H3N2 accessed from GenBank. Bioinformatic analysis was performed with the web-based platform INSaFlu and consensus sequences of each InfA genome were obtained [113] . For comparison, samples obtained at primary healthcare centres in the same region, during the same season, were also included. A phylogenetic tree was constructed using the maximum likelihood method in Mega® Version 7. Bootstrap values were obtained from 500 replicates and displayed on nodes if >70%. The modelling process consisted of the following consecutive steps:
(1) Identifying key variables with a potential influence on in-hospital transmission of influenza.
(2) Construction and technical validation of the model.
(3) Selecting the model scenarios of interest.
5
Amplicon Sequencing Protocol for SELEX
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Using the primers WP20F1 and WP20R1, the PCR product of the SELEX protocol was purified using a MinElute PCR purification kit (Qiagen). The library was sequenced using the AB library builder system (Thermo Fisher Scientific) and amplified according to the protocol for Ion Xpress Plus and Ion Plus library preparation for the AB library builder system. The library was then purified using the Agencourt AMPure XP reagent (Beckman Coulter). The library size and its concentration were assessed using a Bioanalyzer high-sensitivity chip (Agilent Technologies). The samples were pooled, followed by template preparation on the Ion Chef system, using the Ion PI Hi-Q Chef kit (Thermo Fisher Scientific). The samples were then loaded onto Ion PITM v3 chips and sequenced using the Ion Proton system, with the Ion PITM Hi-Q sequencing 200 kit chemistry (Thermo Fisher Scientific).
6
Ion Torrent Sequencing of Poxviral DNA
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After quantification using Qubit dsDNA HS Assay Kit and Qubit 2.0 fluorometer (ThermoFisher Scientific) of purified PCR products from qPCR assays, libraries were built adding barcodes, for sample identification, and primers using the AB Library Builder System (ThermoFisher Scientific). To ensure equimolar pooling of the barcoded samples, a quantification step was included, using the 2100 Bioanalyzer instrument (Agilent Technologies). An emulsion PCR of the pools and loading on-chip was performed using the automated Ion Chef instrument (ThermoFisher). The S5 Ion torrent (Thermo Fisher Scientific) was used for sequencing following the manufacturer's instructions. A de novo contig was produced using CLC genomics workbench software (Qiagen) and a blast was performed to identify the poxviral species amplified.
7
Ion Proton Sequencing of SELEX Products
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The PCR product of the SELEX amplified products using WP20F1 primer was purified with the MinElute® PCR Purification Kit (Qiagen). The sequencing library was conducted using the AB Library Builder™ System (ThermoFisher Scientific) and amplified according to the Ion Xpress™ Plus and Ion Plus Library Preparation for the AB Library Builder™ System protocol. It was then purified using the Agencourt® AMPure® XP reagent (Beckman Coulter). The library size and its concentration were assessed by a Bioanalyzer High Sensitivity Chip (Agilent Technologies). The samples were pooled, followed by a template preparation on the Ion Chef™ System, using the Ion PI Hi-Q Chef Kit (ThermoFisher Scientific). The samples were then loaded on an Ion PI™ v3Chips and sequenced on the Ion Proton™ System, using the Ion PI™ Hi-Q Sequencing 200Kit chemistry (ThermoFisher Scientific).
8
Ion Torrent Sequencing Library Preparation
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Libraries were built from the fragmented PCR products by using the Ion Plus Fragment Library Kit on an AB Library Builder System (ThermoFisher, Waltham, MA USA) according to manufacturer's protocol. The newly built libraries were amplified for eight cycles and purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA). Samples were then further size selected to about 370 bp by using a Pippin Prep (Sage Science, Beverly, MA, USA). The recovered materials were analyzed using the Agilent High Sensitivity D1000 ScreenTape System on a TapeStation 2200 (Agilent, Santa Clara, CA, USA). The DNA concentration was thereafter estimated using an Ion Quantitation Kit (ThermoFisher). The libraries were diluted and pooled to reach a final concentration of 50 pM.
Template preparation and chip loading was performed on the Ion Chef instrument using the Ion PGM™ Hi-Q™ View Chef Kit according to the manual (ThermoFisher). An Ion 318 chip (ThermoFisher) was used, and sequencing was conducted on an Ion Torrent PGM with an Ion PGM Hi-Q View Sequencing Kit (ThermoFisher).
Template preparation and chip loading was performed on the Ion Chef instrument using the Ion PGM™ Hi-Q™ View Chef Kit according to the manual (ThermoFisher). An Ion 318 chip (ThermoFisher) was used, and sequencing was conducted on an Ion Torrent PGM with an Ion PGM Hi-Q View Sequencing Kit (ThermoFisher).
9
Targeted DNA Sequencing Protocol
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The amplicons of each patient were pooled and later fragmented in 200 pb using a Bioruptor ® standard sonication device (Diagenode). Libraries were built using the automated AB Library Builder System (Thermo Fisher Scientific), using Ion ™ Plus Library Kit. To normalizing the number of molecules required for emulsion PCR (ePCR), a quantitation step was performed in LightCycler® 480 Instrument II (Roche Life Science). The ePCR was performed in an automated Ion OneTouch 2 platform (Thermo Fisher Scientific) in accordance with manufacturer protocols. Finally, the mix was loaded on an Ion 318™ Chip Kit-Ion Torrent ™ (Thermo Fisher Scientific) and sequencing reactions were performed on an Ion Personal Genome Machine™ (PGM™) System using Ion PGM™ HI-Q™ View Sequencing (Thermo Fisher Scientific) kit.
10
Whole-Exome Sequencing of Tumor and Normal Tissues
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Tumor and normal tissues were dissected and collected separately from frozen sections under microscopic guidance. DNA was extracted using a ChargeSwitch® gDNA Mini Tissue Kit (Life Technologies, Carlsbad, CA). The extracted DNA was constructed into a fragment library using a SOLiD Fragment Library Construction Kit (Life Technologies) or the AB Library Builder System (Life Technologies). Constructed libraries were subjected to whole-exome enrichment using a SureSelect Human All Exon Kit (Agilent Technologies Inc., Santa Clara, CA) or a TargetSeq™ Target Enrichment Kit (Life Technologies). The prepared exome libraries were sequenced using the massively parallel deep sequencer SOLiD 4 or 5500xl SOLiD System (Life Technologies) using the paired-end sequencing method. Data were analyzed using LifeScope software (Life Technologies) with mapping on the Human Genome Reference, GRCh37/hg19 (The Genome Reference Consortium; http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/index.shtml ). All procedures were performed according to the manufacturers' instructions. Obtained data were annotated and stringently filtered to exclude false variation calls using our previously described programs developed in-house25 (link).
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