We quantified libraries using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and KAPA qPCR, checked for index diversity (File S10 ), and then normalized and pooled all libraries at 10 nM (File S11 ). We also ensured the quality of library pools by running 1 µL on a Bioanalyzer High Sensitivity chip (Agilent Technologies, Santa Clara, CA, USA). We combined the iTru and iNext E. coli library pools (File S1 ) with samples from other experiments, and we sequenced the combined pools using a single run in Illumina MiSeq v2 500 cycle kit (PE250). We combined the eukaryotic libraries with additional TruSeq libraries from other experiments and sequenced these on a separate run of Illumina MiSeq v2 500 cycle kit to produce PE250 reads.
Miseq v2 500 cycle kit
Manufactured by Illumina
Sourced in United States
The MiSeq v2 500 cycle kit is a laboratory equipment product designed for DNA sequencing. It provides the necessary reagents and consumables to perform up to 500 sequencing cycles on the MiSeq platform.
Automatically generated - may contain errors
Lab products found in correlation
16s metagenomic sequencing library preparation guide, by Illumina (4 mentions)
Spriselect beads, by Beckman Coulter (2 mentions)
Miseq instrument, by Illumina (2 mentions)
Dneasy powersoil kit, by Qiagen (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Gel purification kit, by Qiagen (1 mentions)
2100 bioanalyzer dna 1000 chip, by Agilent Technologies (1 mentions)
Fragment analyzer ce, by Agilent Technologies (1 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (1 mentions)
Nextera xt sample preparation kit, by Illumina (1 mentions)
Quant it dsdna high sensitivity assay, by Thermo Fisher Scientific (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Qubit dsdna hs kit, by Thermo Fisher Scientific (1 mentions)
Kapa library quantification kit for, by Illumina (1 mentions)
Agilent d1000 screentape assay, by Agilent Technologies (1 mentions)
19 protocols using miseq v2 500 cycle kit
1
Library Quantification and Pooling for Illumina Sequencing
2
16S rRNA Amplicon Sequencing Protocol
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Each library was prepared according to the “Illumina 16S Metagenomic Sequencing Library Preparation Guide” with a primer set (27Fmod: 5′-AGR GTT TGA TCM TGG CTC AG-3′ and 338R: 5ʹ-TGC TGC CTC CCG TAG GAG T-3ʹ) targeting the V1–V2 region of the 1S rRNA gene. Then, 251 bp paired end sequencing of the amplicon was performed on a MiSeq (Illumina) using a MiSeq v2 500 cycle kit. Paired end sequences were merged using PEAR (http://sco.h-its.org/exelixis/web/software/pear/ ). Merged reads were quality-trimmed with BBtrim (bbmap.sourceforge.net). Twenty thousand reads per sample were randomly selected using random_sequence_sample.pl (ualberta.ca/~stothard/software.html) for further analysis. The processed sequences were clustered into OTUs defined by a 97% similarity cutoff using UCLUST version 1.2.22q. Representative sequences for each OTU were then classified taxonomically by using RDP Classifier version 2.2 and the Greengenes 13_8 database. The bioinformatics pipeline QIIME, version 1.9.1, was used as the informatics environment for all relevant processing of raw sequencing data.
3
Illumina MiSeq Library Preparation
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Equimolar concentrations of PCR amplicons were pooled together and were subsequently gel purified with the Qiagen Gel Purification Kit (Qiagen). Clean PCR products were quantified with the Qubit 4.0 Fluorometer (Life Technologies) and a 2100 Bioanalyzer DNA 1000 chip (Agilent Technologies) before they were shipped on dry ice to Laragen, Inc. (Culver City) for sequencing.
The library was then size verified with the Fragment Analyzer CE (Advanced Analytical Technologies Inc.) and quantified with the Qubit High Sensitivity dsDNA kit (Life Technologies). Finally, the library was denatured and diluted then spiked with 10% PhiX V3 library and loaded on an Illumina MiSeq V2 500 cycle kit for 250 base, paired-end reads.
The library was then size verified with the Fragment Analyzer CE (Advanced Analytical Technologies Inc.) and quantified with the Qubit High Sensitivity dsDNA kit (Life Technologies). Finally, the library was denatured and diluted then spiked with 10% PhiX V3 library and loaded on an Illumina MiSeq V2 500 cycle kit for 250 base, paired-end reads.
4
16S rRNA Metagenomic Sequencing Protocol
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Metagenomic analyses were performed via 16S rRNA sequencing. Each library was prepared according to the ‘Illumina 16S Metagenomic Sequencing Library Preparation Guide’, with a primer set (27Fmod: 5′-AGR GTT TGA TCM TGG CTC AG-3′ and 338R: 5ʹ-TGC TGC CTC CCG TAG GAG T-3ʹ) targeting the V1–V2 region of the 16S rRNA gene. Then, 251 bp paired end sequencing of the amplicon was performed on a MiSeq (Illumina) using an MiSeq v2 500 cycle kit (first PCR: 15 cycles and second PCR: 15 cycles14 (link)). The paired end sequences obtained were merged, filtered and denoised using DADA2. Taxonomic assignment was performed using QIIME2 feature classifier plugin with the Greengenes 13_8 database.15 (link) The bioinformatics pipeline QIIME2 (V.2020.2) was used as the informatics environment for all the processing of raw sequencing data. To visualise the positions of each group, the Emperor software tool was used for principal component analysis (PCA).16 (link)
For identification of the bacterial flora via metagenomic analysis, a positive result was defined as the predominance of a specific bacterial population.
For identification of the bacterial flora via metagenomic analysis, a positive result was defined as the predominance of a specific bacterial population.
5
Nextera XT Amplicon Library Preparation
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Following PCR, the amplicons were purified with 1X AMPure XP beads (Beckman Coulter Inc), quantified with Quant‐iT dsDNA High Sensitivity Assay (Invitrogen), and normalized to 0.2 ng/μL. Indexed paired‐end libraries were generated from 2.5 μL of 0.2 ng/μL amplicon pool using Nextera XT Sample Preparation Kit (Illumina) following the manufacturer's protocol. Amplified libraries were purified using 0.8X AMPure XP beads, quantitated using Quant‐iT dsDNA High Sensitivity Assay (Invitrogen), and evaluated for fragment size in the Agilent 2100 BioAnalyzer System using the Agilent High Sensitivity DNA Kit (Agilent Technologies). Libraries were then diluted to 2 nmol/L in preparation for pooling and denaturation for running on the Illumina MiSeq (Illumina). Pooled libraries were NaOH denatured, diluted to 12.5 pmol/L and sequenced on the Illumina MiSeq using 2 × 250 bp paired‐end reads with the MiSeq v2 500 cycle kit (Illumina). Five percent Phi‐X (Illumina) spike‐in was added to the libraries to increase library diversity by creating a more diverse set of library clusters. Low diversity libraries are common for amplicon pools and occur when a significant number of reads have similar sequences, for example, in amplicon pools.
6
16S rRNA Amplicon Sequencing of Gut Microbiome
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Extraction of bacterial DNA from feces was performed as described previously (42 (link)). Libraries were prepared in accordance with the ‘Illumina 16S Metagenomic Sequencing Library Preparation Guide’ with a primer set (27Fmod: 5′-AGRGTTTGATCMTGGCTCAG-3′ and 338R: 5′-TGCTGCCTCCCGTAGGAGT-3′) targeting the V1–V2 region of the 16S rRNA gene. Then, 251 bp paired-end sequencing of the amplicon was performed on a MiSeq (Illumina) using a MiSeq v2 500 cycle kit. Raw paired-end sequences were merged using PEAR (http://sco.h-its.org/exelixis/web/software/pear/ ), and 50 000 reads per sample were randomly selected using seqtk (https://github.com/lh3/seqtk ) for further analysis. The processed sequences were clustered into operational taxonomic unit (OTU) defined at a 97% similarity cutoff using UCLUST version 1.2.22q. Representative sequences for each OTU were then classified taxonomically using RDP Classifier version 2.2, with the Greengenes 13_8 database. The bioinformatics pipeline QIIME, version 1.9.1, was used as the informatics environment for all relevant processing of sequencing data and calculation of relative bacterial abundances.
7
Single B cell BCR sequencing
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Amplification of BCR heavy and light chains from single sorted B cells was performed by iRepertoire Inc. (Huntsville, AL, USA) as previously described [24 (link)]. Briefly, RT-PCR1 was performed with nested, multiplex primers covering both heavy, kappa, and lambda loci, and including partial Illumina adaptors. After RT-PCR1, the first round PCR1 products were rescued using SPRISelect Beads (Beckman Coulter, Brea, USA). A second PCR was performed with dual-indexed primers that complete the sequencing adaptors introduced during RT-PCR1 and provide plate positional information for the sequenced products. Sequencing was performed using the Illumina MiSeq v2 500-cycle kit with 250 paired-end reads.
8
16S rRNA Amplification and Sequencing
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DNA from swabs was extracted with the DNeasy PowerSoil kit without modifications (Qiagen, Hilden, Germany). The V4 region of 16S rRNA was amplified using 515F and 806 R primers and processed on a MiSeq instrument per Illumina’s 16S protocol (using a Miseq V2–500 cycle kit to generate 2 × 250 paired-end reads) (Illumina, San Diego, CA, USA).55 (link)
9
16S rRNA Sequencing Library Preparation
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A total of 54 samples, including additional experimental controls, were submitted for 16S rRNA sequencing. The Illumina 16S metagenomic Sequencing Library Preparation protocol was followed. All samples underwent amplicon PCR clean-up using AMPure beads (Beckman Coulter, Indianapolis, IN, USA). Illumina sequencing adapters and dual-index barcodes were added to each library using an Index PCR Illumina XT Index Kit v2 (Illumina Inc., San Diego, CA, USA) followed by PCR clean-up with AMPure beads (Beckman Coulter). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (Thermo Fisher Scientific, Waltham, MA, USA). Library fragment-length distributions were analysed using the Agilent TapeStation 4200 and the Agilent D1000 ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA). Libraries were then pooled in equimolar amounts. Library pool quantification was performed using the KAPA Library Quantification Kit for Illumina (Roche). The library pool was sequenced on an Illumina MiSeq using a MiSeq v2 500 cycle Kit (Illumina; MS-102-2003, San Diego, CA, USA) to generate 250 bp paired-end reads.
10
Fecal Microbiome Profiling by 16S rRNA Sequencing
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DNA was extracted from fecal samples using a QIAamp DNA Mini kit (Qiagen, Tokyo, Japan). Each library was prepared according to “Illumina 16S Metagenomic Sequencing Library Preparation Guide” with a primer set (27Fmod:
5ʹ- AGR GTT TGA TCM TGG CTC AG-3ʹ and 338R: 5ʹ-TGC TGC CTC CCG TAG GAG T-3ʹ) targeting the V1–V2 region of the 16S rRNA gene. 250 bp paired end sequencing of the amplicon was performed on a MiSeq (Illumina) using MiSeq
v2 500 cycle kit. The resulting sequences were analyzed using the QIIME pipeline [10 (link)].
For determination of CFU in fecal samples, fecal samples were diluted with a buffer that consisted of 0.45 g KH2PO4 (Kishida Chemical, Osaka, Japan), 1.68 g
Na2HPO4•12H2O (Kanto Chemical, Tokyo, Japan), 0.05 g ʟ-cysteine HCl•H2O, 0.05 g Tween® 80, and 0.1 g agar in 100 ml distilled water. Samples were
subjected to serial 10-fold (w/v) dilutions with dilution buffer and vortexed. In order to calculate the total number of bacteria, 50 µl of the diluted sample was applied to GAM agar medium (Nissui
Pharmaceutical, Tokyo, Japan) and cultured for 48 hr under anaerobic environment. Finally, the number of colonies was counted, and the log CFU/g was calculated.
5ʹ- AGR GTT TGA TCM TGG CTC AG-3ʹ and 338R: 5ʹ-TGC TGC CTC CCG TAG GAG T-3ʹ) targeting the V1–V2 region of the 16S rRNA gene. 250 bp paired end sequencing of the amplicon was performed on a MiSeq (Illumina) using MiSeq
v2 500 cycle kit. The resulting sequences were analyzed using the QIIME pipeline [10 (link)].
For determination of CFU in fecal samples, fecal samples were diluted with a buffer that consisted of 0.45 g KH2PO4 (Kishida Chemical, Osaka, Japan), 1.68 g
Na2HPO4•12H2O (Kanto Chemical, Tokyo, Japan), 0.05 g ʟ-cysteine HCl•H2O, 0.05 g Tween® 80, and 0.1 g agar in 100 ml distilled water. Samples were
subjected to serial 10-fold (w/v) dilutions with dilution buffer and vortexed. In order to calculate the total number of bacteria, 50 µl of the diluted sample was applied to GAM agar medium (Nissui
Pharmaceutical, Tokyo, Japan) and cultured for 48 hr under anaerobic environment. Finally, the number of colonies was counted, and the log CFU/g was calculated.
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