Sequencing of the amplicons was conducted at University of New South Wales (UNSW) using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) with a paired-end 300 base pair sequencing protocol. A pooled library (20 nM) and a PhiX control v3 (20 nM) (Illumina) were mixed with 0.2 N fresh NaOH and HT1 buffer (Illumina) to produce the final concentration at 12 pM each. The resulting library was mixed with the PhiX control v3 (5%, v/v) (Illumina) and 600 μl loaded on a MiSeq1 v2 (500 cycles) Reagent cartridge for sequencing. All sequencing procedures were monitored through the Illumina BaseSpace® website.
Ht1 buffer
Manufactured by Illumina
Sourced in United States
The HT1 buffer is a laboratory solution used to prepare samples for sequencing on Illumina's instruments. It is designed to maintain the integrity of nucleic acid samples during the sequencing process.
Automatically generated - may contain errors
Lab products found in correlation
Phix control v3, by Illumina (7 mentions)
Fragment analyzer ce, by Agilent Technologies (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Miseq platform, by Illumina (4 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (4 mentions)
Miseq instrument, by Illumina (4 mentions)
Phix control v3 library, by Illumina (4 mentions)
Miseq v3 600 cycle kit, by Illumina (3 mentions)
Miseq v3 150 cycle reagent cartridge, by Illumina (3 mentions)
Agilent dna high sensitivity chip, by Agilent Technologies (3 mentions)
Nextera xt dna sample preparation kit, by Illumina (2 mentions)
Miseq reagent kits v2, by Illumina (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Basespace website, by Illumina (2 mentions)
Kapa sybr fast qpcr kit, by Roche (2 mentions)
Eb buffer, by Qiagen (2 mentions)
Agencourt ampure xp pcr purification kit, by Beckman Coulter (2 mentions)
Qubit 2.0 fluorometer, by Beckman Coulter (2 mentions)
Miseq v2 300 cycle kit cassette, by Illumina (2 mentions)
2100 bioanalyzer dna 1000 chip, by Agilent Technologies (1 mentions)
43 protocols using ht1 buffer
1
MiSeq Sequencing of Amplicons
2
Illumina MiSeq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pooled PCR products were gel purified using the Qiagen Gel Purification Kit (Qiagen, Frederick, Maryland, USA). Clean PCR products were quantified using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA), and samples were combined in equimolar amounts. Prior to submission for sequencing, libraries were quality checked using the 2100 Bioanalyzer DNA 1000 chip (Agilent Technologies, Santa Clara, CA). Pooled libraries were stored at −20 °C until they were shipped on dry ice to the California State University (North Ridge, CA) for sequencing. Library pools were size verified using the Fragment Analyzer CE (Advanced Analytical Technologies Inc., Ames IA) and quantified using the Qubit High Sensitivity dsDNA kit (Life Technologies, Carlsbad, CA). After dilution to a final concentration of 1 nM and a 10% spike of PhiX V3 library (Illumina, San Diego CA), pools were denatured for 5 minutes in an equal volume of 0.1 N NaOH, then further diluted to 12 pM in Illumina's HT1 buffer. The denatured and PhiX-spiked 12 pM pool was loaded on an Illumina MiSeq V2 500 cycle kit cassette with 16S rRNA library sequencing primers and set for 250 base, paired-end reads.
3
Metagenomic Sequencing of Environmental Isolates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sample DNA concentration was determined using Qubit high-sensitivity assay. Isolates were diluted to 1 ng 5 µL−1 (0.2 ng µL−1) to be used as input for the Nextera XT DNA sample preparation kit (Illumina). Samples were 'tagmented' and amplified with the associated Nextera XT index kit according to the manufacturer's specifications. Finalized libraries were normalized to 2–4 nmol L−1 and denatured using Nextera XT normalization beads (Illumina) and 0.1 mol L−1 NaOH, respectively. A metagenomic multiplex sample was achieved by pooling 10 µL of each isolate. Twenty-four microliters of the multiplex sample were added to 576 µL HT1 buffer (Illumina) for a final volume of 600 µL and loaded on to a MiSeq V2 cartridge. Sample was sequenced on a MiSeq V2 platform.
4
16S rRNA Gene Sequencing Pipeline
5
Illumina-based Amplicon Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplicon sequencing was performed with the use of the MiSeq platform (Illumina, San Diego, CA, USA). Amplicons were constructed with the use of the NEBNext® DNA Library Prep Master Mix Set for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. Libraries were normalized to equimolar concentrations and quantified with a fluorimetry technique on Qubit 3.0 Fluorometer (Invitrogen, Waltham, MA, USA) using the dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA). The libraries were denatured in the presence of 0,2 N NaOH and diluted with HT1 buffer (Illumina) to a final concentration of 8 pM. Libraries were sequenced on a MiSeq platform (Illumina) using the MiSeq Reagent Kits v2 (Illumina) with the same primers used as in the previous PCR reaction.
6
Shotgun Sequencing of Microbial DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Shotgun DNA sequencing was performed on the Illumina HiSeq platform. DNA was extracted using the Qiagen DNeasy PowerSoil kit and was quantified using the Quant-iT PicoGreen dsDNA assay (Thermo Fisher). DNA sequencing libraries were prepared using one-quarter-scale NexteraXT reactions (Illumina). The resulting DNA libraries were denatured with NaOH, diluted to 8 pM in Illumina’s HT1 buffer, and spiked with 1% PhiX, and a HiSeq 1× 100-cycle v3 kit (Illumina) was used to sequence samples. For the ultradeep shotgun sequencing, 64 separate libraries were prepared as described above but using full Nextera reaction mixtures from a homogenized stool sample and were multiplexed on a HiSeq 3000 high-output run, using an entire run per sample.
7
Sequencing Library Preparation and Denaturation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10 µl of the 2 nM sequencing library was denatured by adding 10 µl of 0.2 N NaOH and incubating at room temperature for 5 minutes, then the library was diluted to 8 pM in Illumina’s HT1 buffer, spiked with 15% PhiX, and sequenced on a portion of a MiSeq 2 × 300 (600 cycle v3) lane.
8
DNA Library Normalization and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were normalized using a SequalPrep capture-resin bead plate (Life Technologies, Carlsbad, CA, USA) and pooled using equal volume. The final pools were quantified via PicoGreen dsDNA assay (Life Technologies, Carlsbad, CA, USA) and diluted to 2 nM. 10 μL of the 2 nM pool was denatured with 10 μL of 0.2 N NaOH, diluted to 8 pM in Illumina’s HT1 buffer, spiked with 15% phiX, heat denatured at 96 °C for 2 min, and sequenced using a MiSeq 600 cycle v3 kit (Illumina, San Diego, CA, USA).
9
Dengue Virus Supernatant Purification and RNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DENV NS1-positive cultures were expanded by passage in 25 ml tissue culture flasks in a volume of 15 ml. Supernatants were harvested and clarified by centrifugation at 1400 rpm for 10 minutes at 4 deg C to remove cellular debris. The clarified supernatant was then transferred to a Millipore centrifugal filter unit (100,000kDa) and centrifuged at 4000 x g for 20 minutes. RNA was extracted from 150–200 μl concentrated virus supernatant using the Roche High Pure RNA isolation kit as per the manufacturer’s instructions. RNA mass/concentration was then determined by fluorescent detection using the Qubit system with Qubit RNA HS Assay kit (Invitrogen). All RNA was stored at -80°C until used. DNA libraries were prepared with the TruSeq Stranded mRNA kit (Illumina), with the use of 200ng of total starting RNA and the exclusion of the polyA selection step. The DNA library sizes were validated with a High Sensitivity DNA kit (Agilent) on the Agilent 2100 Bioanalyzer. The concentration of the libraries were normalised to 4nM using HT1 buffer supplied by Illumina and pooled together. The final concentration of the pooled DNA library was then re-examined using the Qubit ssDNA Assay kit (Invitrogen). The pooled DNA library was sequenced using a MiSeq Reagent Nano Kit, v2 (Illumina).
10
Optimized Nextera XT DNA Library Prep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 ng of DNA was transferred to the library preparation step utilizing the Nextera XT DNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA). The standard manufacturer protocol was modified to optimize library preparation from environmental samples. For each sample, the 20 μl tagmentation reaction contained 5 μl of amplicon tagment mix (ATM), which includes the enzyme used for tagmentation, 10 μl of TD buffer, 1 ng of input DNA (4 μl combined, with or without carrier DNA) and 1 μl of 20 mg/ml bovine serum albumin (BSA). The tagmentation reactions were incubated in a thermal cycler at 50 °C for 5 min. Subsequently, the tagmented DNA was amplified via limited-cycle PCR. The quality and quantity of the purified libraries were assessed using the high-sensitivity (HS) DNA kit on an Agilent 2100 Bioanalyzer. The libraries were normalized to 3 nM, denatured with 0.2 N NaOH for 5 min and diluted 1:100 in HT1 buffer (Illumina) to a final concentration of 15 pM. The diluted libraries were sequenced with an Illumina MiSeq with 50 bp v2 run chemistry. The sequencing length was 60 nt in single-read mode. Each sample was spiked with 0.2 pM phiX174 library (Illumina).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!