Libraries were submitted to the Georgia Genomics and Bioinformatics Core where they were further assessed for quality and concentration using a Fragment Analyzer (Agilent, Santa Clara, CA). Libraries were pooled and sequenced single-end for 75 cycles on one high-output flow cell of an Illumina NextSeq 500 with 5% PhiX control (Illumina) for the first batch and sequenced single-end for 100 cycles on one high-output flow cell of the Illumina NextSeq 2000 with 20% PhiX control added for the second batch for greater base pair diversity. The sequencing run for the first batch generated 225 million reads across samples, with 92% of reads falling above the high-quality threshold of Phred score >30. The second batch generated 525 million reads across samples with 91% at or above the high-quality threshold. Read quality was assessed using FastQC (v0.11.5). Adapters and low-quality sequences (Phred score <25) were trimmed using TrimGalore! (v 0.4.5; --rrbs) and 4bp at the 3’ end was removed due to low overall quality scores of the 3’ end of reads. The efficiency of the bisulfite conversion was determined to be >98% using MethylKit’s (v1.10.0) [59 (link)] conversion statistics in Program R (v3.5.1). Data from the prepared RRBS libraries were made publicly available using NCBI’s Sequence Read Archive (BioProject: PRJNA716946) [60 ].
Phix control
Manufactured by Illumina
Sourced in United States, United Kingdom
PhiX control is a synthetic DNA fragment used for quality control and calibration of DNA sequencing instruments. It serves as a standard reference to evaluate the performance and accuracy of sequencing runs.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (5 mentions)
Miseq system, by Illumina (5 mentions)
Miseq platform, by Illumina (4 mentions)
Qubit dsdna hs assay, by Thermo Fisher Scientific (4 mentions)
Ampure bead, by Beckman Coulter (3 mentions)
Miseq reagent kit v3 600 cycle, by Illumina (3 mentions)
Ampure xp, by Beckman Coulter (3 mentions)
Fragment analyzer, by Agilent Technologies (3 mentions)
Nextera xt index kit, by Illumina (3 mentions)
Miseq reagent kits v2, by Illumina (2 mentions)
Nucleofast 96 pcr plates, by Takara Bio (2 mentions)
Random hexamers, by Integrated DNA Technologies (2 mentions)
Miseq barcodes, by Illumina (2 mentions)
Indexing kit v2 set b, by Illumina (2 mentions)
Superscript 3 rt enzyme, by Thermo Fisher Scientific (2 mentions)
Miseq reagent kit, by Illumina (2 mentions)
Allprep dna rna mini kit, by Qiagen (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Onetaq hot start master mix, by New England Biolabs (2 mentions)
Miseq reagent kit and instrument, by Illumina (2 mentions)
46 protocols using phix control
1
Illumina NextSeq-based RRBS Sequencing
2
HLA Sequencing Library Preparation for Illumina MiSeq
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Amplified DNA for NGS sequencing was prepared according to the supplied instructions (Illumina, Inc., San Diego, CA). Briefly, amplified DNA was purified using magnetic AMPure XP beads, fragmented, and Illumina-specific adaptors were applied using a tagmentation enzyme supplied by Illumina. After tagmentation, fragments were purified using AMPure XP beads and patient-specific indices were added to individual HLA loci by a short PCR, followed by magnetic bead purification. Post-barcoding samples were pooled and the libraries quantified using QuBit fluorimeter. The size of HLA libraries was determined using TapeStation Bioanalyzer 2200.
To assess sequencer-based errors, a 1% to 5% concentration of 12.5 pmol/L PhiX control (Illumina, San Diego, CA) was spiked into pooled HLA libraries. Pooled HLA libraries and PhiX control were loaded onto the cartridge and 2 Â 250 bp sequencing was performed using a regular flow cell on an Illumina MiSeq. Demultiplexing and generation of FASTQ files was performed on the MiSeq system. A total of 34 MiSeq runs were performed and used for quantification of sequencing metrics.
To assess sequencer-based errors, a 1% to 5% concentration of 12.5 pmol/L PhiX control (Illumina, San Diego, CA) was spiked into pooled HLA libraries. Pooled HLA libraries and PhiX control were loaded onto the cartridge and 2 Â 250 bp sequencing was performed using a regular flow cell on an Illumina MiSeq. Demultiplexing and generation of FASTQ files was performed on the MiSeq system. A total of 34 MiSeq runs were performed and used for quantification of sequencing metrics.
3
Illumina Multiplex Sequencing Protocol
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The sample concentration adjustment was based on Qubit® fluorometer (Invitrogen) confirmation on library concentration and quantitative PCR (qPCR) using a 7900HT Fast-Real-Time PCR system. Pooling with the same amount of aliquot from each library was performed for sequencing template preparation. Ten aliquots of equal amounts were pooled together and mixed with Phix Control (Illumina, USA) in a ratio of 3:1; finally, 12.5 pM of product was loaded and sequenced with Illumina platform. Multiplex sequencing reaction was conducted with a MiSeq Reagent Kits v2 with 600 cycles and 2 × 300 bp output on a MiSeq sequencer (Illumina). The total output on reads r1, r2, …,rn fastq datasets was 10 Gb in an average run.
4
Single Cell TCR Sequencing and Analysis
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The sorted T cells were immediately washed with PBS and 0.04% FBS. Single cell barcoding was performed on a Chromium Controller (10X Genomics) according to the manufacturer's protocol. The generating library for paired TCR alpha and beta sequence analysis was processed according to Chromium Single Cell V(D)J reagent kit (10X Genomics). 4 nM of the library was denatured with 0.2 N NaOH and mixed with 1% PhiX control (Illumina). The library sequencing was conducted using a MiSeq system according to the manual (Illumina). Single cell TCR V(D)J sequences were analyzed using the Cell Ranger (v2.0.2) and Loupe V(D)J Browser v2.0 (10X Genomics) software to process multiplexing, barcoding, and gene counting. The dominant TCR paired alpha and beta sequences were codon optimized and designed for the lentiviral construct.
5
16S rRNA Amplicon Sequencing with MiSeq
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Library prepared as previously explained in Inoue et al., 2016 [64 (link)], and deep sequencing were performed using a MiSeq apparatus (Illumina K.K., Tokyo, Japan). Specifically, 341F and 805R primers with 5′ overhang adapter sequences for the second PCR (Polymerase Chain Reaction) were used to amplify the V3–4 region of 16S rRNA genes in each sample. NucleoFast 96 PCR plates (TaKaRa bio, Shiga, Japan) were used to purify the amplicons, and a unique combination of dual indices (I5 and I7 indices) was attached in the second round of PCR. After purification using a SequelPrep Normalization Plate Kit (Thermo Fisher, Tokyo, Japan), the concentration of each sample was normalized. Next, the samples were pooled and concentrated using AMPure XP beads (Beckman Coulter, Tokyo, Japan). Through the SequelPrep Normalization Plate Kit (Thermo Fisher, Tokyo, Japan), ten pM of the library combined with 20% phiX Control (Illumina) was sequenced with 285 bp paired-end bases on MiSeq.
6
Profiling Immune Repertoire Dynamics
7
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.
8
Reduced Representation Bisulfite Sequencing
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Reduced representation bisulphite sequencing libraries were constructed with 200 ng input DNA from each sample (n = 24; Table 1 ) using the Premium RRBS Kit (Diagenode, Cat. No. C02030032) according to the manufacturer’s instructions. Unmethylated and methylated spike‐in control sequences are included within the commercial kit to allow for assessment of bisulphite conversion efficiency, which in this experiment exceeded 99% for all samples (mean ± SD = 99.57 ± 0.25). Size selection and clean‐up steps were performed with AMPure XP Beads (Beckman Coulter), and bisulphite‐converted libraries were subjected to 13–15 amplification cycles in the final PCR (polymerase chain reaction). Resulting libraries were stored at −80°C prior to submission to the Georgia Genomics and Bioinformatics Core (GGBC; University of Georgia, Athens, GA) for sequencing. Quality of the libraries was assessed using an Agilent Fragment Analyzer, and pooled libraries were sequenced on a single Illumina NextSeq 500 High Output flowcell (75 bp, single‐end) with 20% Illumina PhiX control. Sequencing generated a total of 300 million reads, with 94% meeting or exceeding the Phred score quality threshold of >30. Raw reads were assessed for quality using fastqc (version 0.11.8; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) and multiqc (version 1.8; Ewels et al., 2016 (link)).
9
Illumina Stranded Total RNA Sequencing of Mouse Placenta
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Libraries were prepared with the Illumina TruSeq Stranded Total RNA Library Preparation kit with Ribo Zero Gold (Illumina, #RS-122-2301) according to the instructions, using 1000 ng total mouse placenta RNA as starting material. Libraries were quantified on a Qubit fluorometer and by qPCR with a KAPA Library Quantification Kit for Illumina libraries (# KK4828). Size distribution was checked using the Agilent High Sensitivity DNA Assay (#5067-4626) on an Agilent Bioanalyzer. Samples were denatured using 1N NaOH, diluted to a concentration of 3 nM with ExAMP mastermix and loaded onto a HiSeq 4000 machine (Illumina, San Diego, CA; #SY-401-4001) with 1% PhiX control (Illumina, #FC-110-3001) spiked in. HiSeq 3000/4000 flow cells and HiSeq 3000/4000 SBS sequencing chemistry were used for paired-end sequencing with a read length of 100 bp for each direction. Sequencing was performed at the Helmholtz Center (Munich, Germany).
10
HPV DNA Amplification and Illumina Sequencing
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HPV DNA was amplified by PCR using modified GP5+/GP6+ primer pairs extended with overhang adapter sequences required to bind the Illumina® indexes and sequencing [19 (link)]. PCR was performed in 50 μL reaction mixtures containing 1X High-Fidelity Platinum buffer, 3.5 mM MgCl2, 0.6 μM forward and reverse primers and 1U of proofreading Taq polymerase (Platinum™ Taq DNA Polymerase, Thermo Fisher®). The cycling protocol consisted of an activation step (15 min at 95 °C), 21 cycles (1 min at 94 °C and 2 min at 50 °C, decreased by 0.5 °C per cycle to 40 °C and 1.5 min at 72 °C), 24 cycles (1 min at 94 °C, 2 min at 40 °C, 1.5 min at 72 °C), and a final elongation step (4 min at 72 °C) [20 (link)]. Each PCR product was dual-indexed using the KAPA HiFi HotStart Uracil + ReadyMix® (Roche Diagnostics®). The Agencourt Ampure XP beads system (Beckman Coulter®) was used to purify the DNA libraries. The concentrations of the DNA libraries were normalised prior to pooling and sequencing to ensure equal representation of each sample. A PhiX Control (Illumina®) spike-in at 5% was used as an internal control to monitor sequencing quality. The combined library and the PhiX Controls were loaded at 8 pM final concentration on MiSeq using the v3 reagent kit and sequenced by Illumina MiSeq 2 × 300-bp paired-end sequencing.
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