Miseq v3 kit

Stool samples were collected after the end of RBX treatment. DNA was isolated according to the instruction of Fast DNA Spin kit (MoBio, Carlsbad, CA, USA). The quality of the DNA was determined in a NanoDropOne spectrophotometer (Thermo Scientific, Waltham, MA, USA), and DNA purity was analyzed by agarose gel electrophoresis. The 16S rRNA gene primers used targeted the V3-V4 region of the gene. Amplicons were prepared in triplicate, pooled, and quantified. Samples were sequenced on the MiSeq sequencing platform (Illumina, San Diego, CA, USA) using a Miseq V3 kit, following standard Illumina sequencing protocols [15 (link)].

The molarity of the individual libraries was estimated from the lengths and intensity of peaks in the fluorescent quality traces and the concentration of each library measured with the Qubit fluorometer (Life Technologies). All libraries were mixed to have the same final molar concentration and sequenced with the Illumina MiSeq v3 kit or HiSeq 2500 rapid run using 2×35 bp paired end reads. Adapter trimming was turned off during Illumina post-sequencing processing.

For ultra-deep sequencing (UDS) amplicons obtained from pre-treatment samples from four individuals who experienced virological failure were sent to Inqaba Biotechnical industries in Pretoria South Africa for sequencing up to 1 % prevalence. Additional samples from three individuals who virally suppressed and one sample from an individual who experienced low level viremia were also deep sequenced to provide comparison.
Briefly, cDNA samples were fragmented using an ultrasonication approach and the resulting fragments were purified and size selected, end repaired and an illumina specific adapter sequence ligated. Following quantification, the samples were individually indexed and a second size selection step was performed using AMPure XP Beads. Libraries were quality controlled on a DNA chip (Agilent 2100 Bioanalyzer) and then sequenced on illumina’s MiSeq platform, using a MiSeq v3 kit according to the manufacturer’s protocol. Fifty (50) Mb of data (2 × 300bp long paired end reads) were produced for each sample. This was followed by HIV sequence analysis and quality check, genotyping and drug resistance interpretations using Deepchek 1.4 HIV analysis software. A minimum of 461 sequences was required for 99 % confidence at 1 % threshold and a Q30 score measure was applied for basecalling.

Individualized germline IGHV gene databases from NHP MCM01, MCM02, MCM03, MCM04, MCM05, and MCM06 were produced from samples obtained before immunization. In brief, IgM libraries for deep sequencing of antibody repertoires were generated with 5′ multiplex PCR from total PBMC mRNA^{53 (link)}. The mRNA was reverse transcribed with IgM constant region-specific primer containing a unique molecular identifier and a universal outer primer sequence. The cDNA was amplified utilizing the universal 3′ primer and two primer sets covering all gene families and targeting leader and UTR respectively that yield two libraries for each animal. Illumina indices and adapters were introduced by PCR prior to sequencing the library with Illumina’s MiSeq v3 kit. The output library was analyzed with IgDiscover to infer the germline gene IGHV alleles^{31 (link)}. The IGHV database utilized as an input database for the IgDiscover analysis was obtained from genomic DNA sequencing^{54 (link)}, with the addition of non-located (NL) alleles inferred from a larger set of NHPs^{53 (link)}. The heavy chain sequences of the isolated mAbs were assigned to the IGHV individualized database from the respective NHP and a comprehensive IGHJ database obtained from a larger group of NHPs, whereas the light chain was assigned to the IMGT’s light chain database using IgBlast to obtain the SHM and CDR3 sequences.

16S rRNA gene sequencing was performed using a MiSeq V3 kit as per the manufacturer's protocol (Illumina, San Diego, CA, USA). Briefly, the V3-V4 region of the bacterial 16S rDNA was amplified using PCR with forward and reverse primers (5′-TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGA GGC WGC AG-3′ and 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′) with the TaKaRa Ex Taq HS Kit (TaKaRa Bio, Shiga, Japan) from 5 ng of DNA from faecal samples. After the PCR products were purified with Agencourt AMPure XP (Beckman Coulter, Indianapolis, IN, USA) and amplified using a Nextera XT Index Kit v2 (Illumina, San Diego, CA, USA). After the second round of PCR, the products were again purified using Agencourt AMPure XP. The library was quantified, normalised, and pooled in equimolar amounts. Sequencing was conducted using a paired-end 2 × 300-bp cycle run on an Illumina MiSeq system with a MiSeq Reagent Kit v.3 (600 cycles).