Half of the cDNA from the 20 µl reverse transcription reaction from bio–tREX was added into a PCR mix containing 25 µl Q5 High-Fidelity 2x Master Mix, 12 µl water and 2 µl of a 10 µM predefined mix of indexing primers (see Supplementary Data 2 ). A standard PCR program with 29 amplification cycles and an annealing temperature of 60 °C was used. Extension times were adapted to the amplicon length according to the manufacturer’s guidelines. DNA was bound to 100 µl of Agencourt AMPure XP (Beckman) for 10 min and the beads were washed 3 times with 200 µl 80% ethanol. Beads were dried and DNA was eluted in 25 µl water. DNA concentrations were measured using Qubit 2 Fluorometer (Life Technologies) and the Qubit 1x dsDNA HS Assay Kit (Invitrogen) and 80 ng of each amplicon were combined into the NGS library. The combined library was diluted in HT1 Hybridization Buffer (Illumina) to a concentration of 2 nM. PhiX (Illumina) was added to increase the diversity of the library at a 20% molar ratio. 12 µl of the library was added to 18 µl HT1 Hybridization Buffer (Illumina) and 20 µl of the diluted mixture was used for NGS analysis.
Ht1 hybridization buffer
Manufactured by Illumina
Sourced in United States
The HT1 Hybridization Buffer is a laboratory reagent used in various genomic and molecular biology applications. It is designed to facilitate the hybridization process, which is a crucial step in many experimental procedures. The buffer helps to create the optimal conditions for the specific binding of nucleic acid molecules, such as DNA or RNA, to their complementary sequences. Its core function is to provide a suitable environment for efficient and reliable hybridization, supporting the fundamental steps of various genomic analysis techniques.
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Lab products found in correlation
Phix control v3, by Illumina (2 mentions)
Miseq instrument, by Illumina (2 mentions)
Agencourt ampure xp, by Beckman Coulter (1 mentions)
Qubit 1x dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer 2, by Thermo Fisher Scientific (1 mentions)
Basespace application, by Illumina (1 mentions)
Miseq v2 500 cycle reagent cartridge, by Illumina (1 mentions)
Miseq reporter, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Sodium hydroxide (naoh), by Illumina (1 mentions)
Phix control, by Illumina (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Miseq reagent kit v2 300, by Illumina (1 mentions)
Lightcycler lc480 2, by Roche (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
Qubit dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2 500, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
11 protocols using ht1 hybridization buffer
1
Amplicon Sequencing Library Preparation
2
Paired-end Sequencing on Illumina MiSeq Platform
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A pooled library (20 nM) and a PhiX control v3 (20 nM) (Illumina) were mixed with 0.2 N fresh NaOH and hybridization buffer HT1 (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 MiSeq® v2 (500 cycle) Reagent cartridge for obtaining a paired-end 2 × 250 bp sequencing. All sequencing procedures were monitored through the Illumina BaseSpace® application. Demultiplexed FASTQ files were generated by Illumina MiSeq Reporter and a total of 2.5 Gbases were obtained (average of 286,167 reads per sample). Sequencing reads are available in NCBI Short Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra ) under ID PRJNA355083.
3
Optimized Pooled Library Sequencing
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To achieve optimal cluster density, equal library volumes (5 μl) of individual libraries were pooled into a sterile 1.5 ml tube and the pool was quantified in triplicate by Qubit fluorometric quantitation with the Qubit dsDNA HS Assay Kit before sequencing. It was assumed that the library size was 600 bp. The pooled libraries were diluted in resuspension buffer (RSB; Illumina) to give a final starting concentration of 4 nM. Libraries were denatured in NaOH and were further diluted in Hybridization Buffer (HT1; Illumina) from 4 nM to 20 pM and diluted again in HT1 to give a final loading concentration of approximately 10–14 pM. The sequencing was performed on a MiSeq platform (Illumina) using v3 chemistry, as 300‐cycle paired‐end runs. The pool was spiked with 1% PhiX, loaded at 20 pM and an average of 32 samples were loaded per MiSeq flow cell. The isolates included in this study were distributed over four sequencing runs.
4
Library Preparation and Sequencing Protocol
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DNA insert fragments in the library were tested using the Agilent 2100 Bioanalyzer (Agilent 1000 Reagents; Agilent Technologies, Inc., Santa Clara, CA, USA), and the concentration was quantified using an ABI StepOnePlus Real-Time PCR System (TaqMan Probe; Applied Biosystems; Thermo Fisher Scientific, Inc.). The aforementioned library was qualified by NaOH according to the protocol (Illumina, Inc., San Diego, CA, USA), diluted with hybridization buffer HT1 (Illumina, Inc.) to a final dilution of 10 pm, then subjected to flowcell hybridization (15 min at 95°C, 30 cycles of 30 sec at 94°C, 30 sec at 59°C and 1 min at 72°C, followed by a final extension cycle of 10 min at 72°C) and then combined with TruSeq PE Cluster kit v3-cBot-HS reagent (Illumina, Inc.) to complete bridge PCR amplification, according to the manufacturer's protocol. Finally, the prepared flowcell was tested using the MiSeq sequencing.
5
Illumina Sequencing of Amplicons
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Amplicons were sequenced through one single end sequencing reads of 50 nucleotides. Libraries were denatured and loaded onto Illumina 50-cycle V2 cartridges, according to the manufacturer's suggested workflow. Briefly, libraries were combined at a concentration 4 nM and denatured for 5 min at room temperature with freshly prepared 0.2N NaOH. After incubation, the reaction was neutralized with ice-cold, HT1 Hybridization Buffer (Illumina) diluted to 12 pM and loaded onto the cassette. Illumina PhiX Control was used as internal quality control. PhiX was denatured as mentioned above and diluted in buffer HT1 to a concentration of 12.5 pM. PhiX was added to denatured, diluted editing amplicon libraries to a concentration final concentration of 1% of the combined library volume.
6
Next-Generation Sequencing Library Pooling
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All libraries for NGS were diluted to a 4 nM concentration, based on their determined concentrations and fragment sizes. Equal volumes of 5 μl of each library were pooled and denatured with NaOH (0.2 N final concentration) for 5 min. The pooled mixture was vortexed and spun briefly and incubated at room temperature for 5 min. The pool was further diluted to 20 pM concentration with chilled HT1 hybridization buffer (Illumina, USA). Using the same buffer, the final concentration of the library pool was diluted to 10 pM. Control library (3% PhiX library, Illumina, USA) was added and the pool was snap-chilled on ice. The library pool (600 μl) was loaded in the flow cell of the 500 cycle MiSeq Reagent Kit v2 (Illumina, USA) and pair-end sequencing (2 × 250 bp) was performed on the Illumina MiSeq instrument (Illumina, USA). After automated cluster generation in MiSeq, the sequencing reads were processed and all statistical data generated by the instrument were collected and summarized.
7
Illumina MiSeq Denatured Pooled Sequencing
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Pooled sequencing libraries were denatured with 0.1 M NaOH, diluted to 12 pM with HT1 hybridization buffer (Illumina) and mixed with 40% PhiX Control v3 (Illumina) sequencing control library. Denatured sequencing pools were loaded onto MiSeq Reagent kit V2-300 (Illumina) and 2 × 70 bp sequence reads were generated with an Illumina MiSeq instrument with custom read 1, read 2 and index 1 sequencing primers spiked in the appropriate cartridge positions (12, 14 and 13, respectively) at a final concentration of 0.5 µM.
8
Next-Generation Sequencing Library Quantification
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The quantity and quality of each amplicon pool was assessed by Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific), Bioanalyzer High Sensitivity DNA Kit (Agilent), and qPCR using the KAPA Library Quantification Kit (Roche) on a Light Cycler LC480II (Roche) according to the manufacturer’s instructions. qPCR data were used to calculate sample molarity. 5 μl of the final pool was denatured for 5 min at room temperature using 5 μl of freshly diluted 0.1 N sodium hydroxide (Illumina). The reaction was subsequently terminated by the addition of HT1 hybridization buffer (Illumina) and the library diluted post-denaturation to a final loading concentration of 7–8.5 pM. Libraries were sequenced with 10–20% PhiX (Illumina) using the 2 × 300 bp paired-end protocol.
9
Illumina Paired-End Sequencing Protocol
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Amplicon and whole-genome libraries were sequenced on the Illumina MiSeq system using the MiSeq Reagent Kit v2-500 and v3-600, respectively (Illumina). Amplicon and whole-genome samples that passed quality control were pooled in a 4 nM library with nuclease-free water. 5 μl of the 4 nM library pool was denatured with 5 μl 0.2 N of NaOH and diluted using the HT1 Hybridization Buffer (Illumina) to a concentration of 8 pM for amplicon samples and 10 pm for whole-genome samples. A PhiX library was prepared similarly and added at 30% of the input for amplicon sequencing and 1% for whole-genome libraries. Samples were loaded on the respective MiSeq cartridge and paired-end sequencing reads were generated (Illumina).
10
Illumina Library Denaturation and Sequencing
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Libraries were denatured and loaded onto Illumina 600-cycle V3 cartridges, according to the manufacturer's suggested workflow. Briefly, libraries were combined at equimolar concentrations, denatured and neutralized according to the manufacturer's protocol for 5 min at room temperature with freshly prepared 0.2 N NaOH. After incubation, the reaction was diluted with ice-cold, HT1 Hybridization Buffer (Illumina, San Diego, CA, USA). Illumina PhiX Control (12.5 pm ) was used as an internal quality control according to the manufacturer's protocol at a concentration of 5% of the final, combined library volume.
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