For sequencing, libraries were pooled two-by-two in equimolar amounts (final concentration of 20 pM). Emulsion PCR and enrichment for positive Ion Sphere Particles (ISPs) was performed on the Ion OneTouch 2 and ES enrichment modules, respectively, using the Ion PGM Template OT2 400 Kit (Life Technologies), and sequenced on the Ion Torrent PGM, using the Ion PGM Sequencing 400 Kit and Ion 318 v2 Chips (Life Technologies), according to the manufacturer's protocols. ATLAS-seq sequencing statistics are summarized in Supplementary file 2 .
Ion 318 v2 chip
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain
The Ion 318 v2 Chip is a semiconductor-based sequencing microchip designed for the Ion GeneStudio S5 System. It is capable of generating high-quality sequencing data for a wide range of applications, including targeted sequencing, whole-exome sequencing, and small-genome sequencing. The chip features a high-density array of semiconductor-based sensors that enable massively parallel sequencing of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Ion pgm hi q sequencing kit, by Thermo Fisher Scientific (8 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (8 mentions)
Ion pgm sequencing 400 kit, by Thermo Fisher Scientific (6 mentions)
Ion pgm sequencing 200 kit v2, by Thermo Fisher Scientific (6 mentions)
Ion pgm template ot2 200 kit, by Thermo Fisher Scientific (6 mentions)
Ion onetouch 2 system, by Thermo Fisher Scientific (6 mentions)
Ion torrent pgm, by Thermo Fisher Scientific (6 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (6 mentions)
Ion pgm template ot2 400 kit, by Thermo Fisher Scientific (5 mentions)
Ion pgm hi q view sequencing kit, by Thermo Fisher Scientific (5 mentions)
Ion pgm system, by Thermo Fisher Scientific (5 mentions)
Ion chef system, by Thermo Fisher Scientific (5 mentions)
Ion pgm, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Ion pgm sequencer, by Thermo Fisher Scientific (4 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (3 mentions)
Kapa library quantification kit, by Roche (3 mentions)
Ion pgm hi q chef kit, by Thermo Fisher Scientific (3 mentions)
Ion pgm hi q ot2 kit, by Thermo Fisher Scientific (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
47 protocols using ion 318 v2 chip
1
Sequencing Using Ion Torrent PGM
2
Targeted Genomic Sequencing Protocol
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Capture of the target regions was performed with reagents from a custom design HaloPlex Target Enrichment kit (Agilent Technologies), according to the HaloPlex Target Enrichment System Protocol. Briefly, the protocol consisted of the following steps: (1) digestion of gDNA with restriction enzymes; (2) hybridization of fragments to probes whose ends are complementary to the target fragments (during this step, fragments are circularized and sequencing and barcode adapters are incorporated); (3) capture of target DNA using streptavidin beads and ligation of circularized fragments; and (4) PCR amplification of captured target libraries.
Quality control of all libraries was performed on the Agilent Bioanalyzer using a High Sensitivity chip. Template dilutions were calculated after library concentrations were normalized to ~100 pM using the Ion Library Equalizer kit (Life Technologies). Library templates were clonally amplified using the Ion One Touch 2™, following the manufacturers’ protocol. Recovered template-positive ion sphere particles (ISPs) were subjected to enrichment according to the manufacturer’s protocol. Samples were subjected to the standard Ion PGM 200 Sequencing v2 protocol using Ion 318 v2 chips (Life Technologies). Up to three samples were loaded per Ion 318 v2 chip due to variable coverage uniformity.
Quality control of all libraries was performed on the Agilent Bioanalyzer using a High Sensitivity chip. Template dilutions were calculated after library concentrations were normalized to ~100 pM using the Ion Library Equalizer kit (Life Technologies). Library templates were clonally amplified using the Ion One Touch 2™, following the manufacturers’ protocol. Recovered template-positive ion sphere particles (ISPs) were subjected to enrichment according to the manufacturer’s protocol. Samples were subjected to the standard Ion PGM 200 Sequencing v2 protocol using Ion 318 v2 chips (Life Technologies). Up to three samples were loaded per Ion 318 v2 chip due to variable coverage uniformity.
3
Small RNA Sequencing via Ion-Torrent
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Small RNA library preparation was performed using the Ion Total RNA-Seq kit v2 (Life Technologies) for both EV samples. For each individual library, RNA was ligated to adapters containing a unique index barcode (Ion Xpress™ RNA-Seq Barcode 1–16 Kit, Life Technologies) to allow libraries to be pooled during Ion-Torrent sequencing (Life Technologies). All libraries were constructed according to manufacturer’s protocol. Briefly, RNA samples were reverse transcribed to cDNA using adapter-specific primers. Using the Magnetic Bead Purification Module (Life Technologies), cDNA samples were size-selected from 94 to 200 nt (the length of the small RNA insert including the 3′ and 5′ adapters). PCR amplification was then performed followed by a library clean-up step using nucleic acid beads (Life Technologies). The quality and quantity of each library were determined by Agilent 2100 Bioanalyser using High Sensitivity DNA kit (Agilent Technologies). Equally pooled libraries were clonally amplified onto Ion Sphere™ Particles (ISPs) supplied by the Ion PGM™ Template OT2 200 kit (Life Technologies). ISP templates were produced by using the OneTouch™ 2 Instrument and enrichment system (Life Technologies). ISPs loaded with libraries were sequenced on the Ion-Torrent PGM™ using Ion™ 318 v2 chips (Life Technologies) and the Ion PGM™ 200 Sequencing Kit v2 (Life Technologies).
4
Ion Torrent PGM Sequencing Protocol
5
Ion Torrent PGM Sequencing of Barcoded Samples
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All barcoded samples were sequenced using the Ion PGM™ Hi-Q™ Sequencing Kit (Life Technologies) in an Ion Torrent PGM instrument (Life Technologies) with Ion 318™ v2 chips (Life Technologies).
Chip loading procedure was performed according to the user guide for the Ion PGM™ Hi-Q™ Sequencing Kit (Life Technologies). A maximum of 16 samples were loaded on a single chip per sequencing run.
Chip loading procedure was performed according to the user guide for the Ion PGM™ Hi-Q™ Sequencing Kit (Life Technologies). A maximum of 16 samples were loaded on a single chip per sequencing run.
6
16S rRNA Amplicon Sequencing Protocol
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DNA was extracted from each newly collected sample using the bead-beating method [16 (link)], and the V1–V2 regions of the 16S rRNA gene were amplified using the following primers: 8F (5-AGA GTT TGA TYM TGG CTC AG-3 ) with the Ion Torrent adapter A and the sample-specific 8-base tag sequence and 338R (5-TGC TGC CTC CCG TAG GAG T-3 ) with the Ion Torrent trP1 adapter sequence. PCR amplification, purification, and quantification of each PCR amplicon was performed as previously described [17 (link)]. The purified PCR amplicons were pooled, and gel purification was performed using the Wizard SV Gel and PCR Clean-Up System (Promega, WI, USA). The DNA concentration was determined using a KAPA Library Quantification Kit (KAPA Biosystems, MA, USA), and the DNA was diluted for use as the template DNA in emulsion PCR. Emulsion PCR and enrichment of template-positive particles were performed using Ion PGM Template Hi-Q View OT2 Kit (Thermo Fisher Scientific) in Ion One Touch 2 system (Thermo Fisher Scientific). The enriched particle was loaded onto Ion 318 v2 chips (Thermo Fisher Scientific), and sequencing was performed on the Ion PGM (Thermo Fisher Scientific) using the Ion PGM Hi-Q view Sequencing Kit (Thermo Fisher Scientific).
7
Detecting Mosaic Variants via PASM
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Personal Genome Machine amplicon seuencing of mosaicism (PASM)
was used to validate candidate mosaic variants that were not located on
mutant hotspots. Targeted PCR amplification was used to capture a region of
400 base pairs around the candidate genome position. The amount of DNA used
for amplification was 20 ng (approximately 6000 genome copies). The primers
are listed in TableS4 . An
amplicon library was prepared using an Ion
XpressTM Plus Fragment Library Kit and
sequenced on an Ion Torrent Personal Genome Machine (PGM) using Ion 318 V2
chips (ThermoFisher). The average read depth for PASM was approximately
12,000×. A hierarchical Bayesian model was used to calculate MAFs with
maximum a posteriori estimation and 95% Bayesian confidence intervals (CIs) (https://github.com/Yyx2626/yyxMosaicHunter ). The MAF threshold for the lower bound of the reference
homozygous variants was 0.5%. The MAF threshold for the heterozygous
variants was 40.0–60.0%. According to our previous benchmark tests, the
validation sensitivity is 0.85, while the specificity is 0.92 (ref.
25 (link)).
was used to validate candidate mosaic variants that were not located on
mutant hotspots. Targeted PCR amplification was used to capture a region of
400 base pairs around the candidate genome position. The amount of DNA used
for amplification was 20 ng (approximately 6000 genome copies). The primers
are listed in Table
amplicon library was prepared using an Ion
XpressTM Plus Fragment Library Kit and
sequenced on an Ion Torrent Personal Genome Machine (PGM) using Ion 318 V2
chips (ThermoFisher). The average read depth for PASM was approximately
12,000×. A hierarchical Bayesian model was used to calculate MAFs with
maximum a posteriori estimation and 95% Bayesian confidence intervals (CIs) (
homozygous variants was 0.5%. The MAF threshold for the heterozygous
variants was 40.0–60.0%. According to our previous benchmark tests, the
validation sensitivity is 0.85, while the specificity is 0.92 (ref.
25 (link)).
8
Ion Torrent PGM Sequencing Protocol
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Libraries were normalized and pooled to an equimolar concentration as recommended by the manufacturer. The pooled libraries was used to generate template-positive Ion Sphere Particles containing clonally amplified DNA. Emulsion PCR was performed on the Ion OneTouch 2 instrument with the Ion PGM Template OT2 200 Kit according the template preparation protocol provided by Ion Torrent (Thermo Fisher). Template-positive Ion Sphere Particles were enriched using the Ion OneTouch Enrichment System and subsequently loaded onto Ion 318 v2 Chips (Thermo Fisher). Sequencing was performed using the Ion PGM System with the Ion PGM Sequencing 200 Kit v2 following the recommended protocol (Thermo Fisher).
9
16S Bacterial Metagenomics Sequencing Analysis
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Sequencing was performed with the use of an Ion Torrent Personal Genome Machine (PGM) platform (Life Technologies, Carlsbad, CA, USA) and 16S Metagenomics Kit (Life Technologies; Carlsbad, CA, USA) as described previously [90 (link)]. A maximum of 26 barcoded 16S samples were sequenced on an Ion 318 v2 chip (Life Technologies, Carlsbad, CA, USA) using the Ion PGM Sequencing 400 Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Sequencing data were analyzed and bacterial taxonomy was evaluated with the use Ion Reporter software (Thermo Fischer, Waltham, MA, USA) and fallowing databases: Curated MicroSEQ(R) 16S Reference Library v2013.1; Curated Greengenes v13.5. Data are presented as a percentage of bacterial taxa in each sample.
Sequencing data were analyzed and bacterial taxonomy was evaluated with the use Ion Reporter software (Thermo Fischer, Waltham, MA, USA) and fallowing databases: Curated MicroSEQ(R) 16S Reference Library v2013.1; Curated Greengenes v13.5. Data are presented as a percentage of bacterial taxa in each sample.
10
16S rRNA Amplicon Sequencing of Soil Microbiome
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DNA was extracted using FastDNA SPIN Kit for Soil (MP Biomedicals, USA). Polymerase chain reaction (PCR) of 16S rRNA V3-4 region was performed employing Probio_Uni_F and Probio_Uni_R [26] (link) primers tagged with 10-11bp unique barcode labels along with the adapter sequence. PCR reaction was carried out using Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific, USA) and GeneAmp® PCR System 9700 (Thermo Fisher Scientific, USA) according to manufacturers' guidelines. Thermal conditions of the PCR reaction were set as follows: 98 °C for 30 seconds, 35 cycles of 98 °C for 10 seconds, 67 °C for 15 seconds, 72 °C for 15 seconds with a final extension at 72 °C for 7 minutes. PCR products were purified using NucleoMag® NGS Clean-Up and Size Select Kit (Macherey-Nagel, Germany). The quality and quantity of amplicons were assessed using Agilent High Sensitivity DNA kit and Agilent 2100 BioAnalyzer (Agilent Technologies, USA). Samples were diluted to 12 pM and pooled. Samples were sequenced on Ion Torrent Personal Genome Machine sequencing platform employing Ion PGM™ Hi-Q™ View OT2 kit (Life Technologies, USA) for template generation and Ion PGM™ Hi-Q™ View Sequencing kit (Life Technologies, USA) on Ion 318 v2 chip (Life Technologies, USA).
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