Nextera xt index kit

Fecal concentrations of EDP1066 for stool persistence and prevalence and the gut microbiome were measured by Diversigen Inc. (Houston, TX, USA) using validated bioanalytical assay methods. In short, fecal microbial DNA was extracted based on the Zymo Research (Irvine, CA, USA) fecal DNA extraction methodology. EDP1066-specific primers and probes had been developed to enable the detection of the L. lactis spp. cremoris strain. The fecal samples were analyzed using a qPCR with a lower limit of quantification of 5.0 copies/5 ng DNA. For gut microbiome analyses, extracted DNA was prepared for Illumina sequencing via PCR amplification of the variable region 4 of the bacterial 16S rRNA gene. After PCR purification using AMPure XP beads (Beckman Coulter Life Sciences, Indianapolis, IN, USA), sample-specific barcodes using Illumina Nextera XT Index kit (Illumina Inc., San Diego, CA, USA) were appended to the PCR products during a second PCR. The PCR products were purified for a second time, and lastly, the PCR products were equimolarly pooled and sequenced on the Illumina MiSeq platform using the MiSeq v3 sequencing kit.

Libraries were prepared from 5 μg of genomic DNA of each sample. The DNA was processed by Illumina Nextera XT sample preparation kit (Illumina Inc. San Diego, CA, USA) and uniquely indexed using Illumina Nextera XT Index Kit (Illumina Inc. San Diego, CA, USA). The libraries were purified and normalized using Agencourt AMPure XP beads (Beckman Coulter, Beverly, Massachusetts). All recovered elutes were pooled in equal volumes. DNA concentration of the pool was determined using Qubit ds DNA HS Assay (Thermo Fisher Scientific Inc. Wilmington, Delaware USA). Paired end reads were generated on Illumina MiSeq platform (Illumina, San Diego, CA, USA) using a paired-end 2 × 250 bp protocol.

DNA was extracted from saliva using the Mo Bio Ultraclean kit (Qiagen) and from dental plaque using the MasterPure Gram-Positive Purification kit with an 18-h incubation. DNA from stool samples was extracted using the MagAttract PowerSoil DNA kit on an Eppendorf epMotion 5075 Liquid Handling Workstation.
(iii) Library preparation and sequencing. For bacterial sequencing, we amplified the V3-V4 regions of the 16S rRNA gene using standard primers (48 (link)) with Illumina Nextera adaptors (Illumina). We profiled fungal communities using the ITS1 region (49 (link)), amplified using published primers (forward primer ITS1F and reverse primer ITS2R) (50 (link)) with adaptors for Illumina multiplexing and an adapted cycling protocol (51 (link)). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter) and multiplexed using the Illumina Nextera XT Index kit. Indexed 16S rRNA and ITS libraries were purified using AMPure XP beads and quantified using the Quant-iT Broad Range dsDNA assay kit (Thermo Fisher Scientific). Libraries were normalized and pooled with a 10% PhiX spike and sequenced on an Illumina MiSeq with a v3 kit. Negative controls were included on all sequencing runs, and a Candida glabrata positive control was included in ITS amplification and sequencing.

The same PCR products obtained previously for the Sanger sequencing of the NS3/4 A, NS5A and NS5B regions were analyzed using a next generation sequencing (NGS) approach. Samples from 15 DAA failing patients as well as 15 untreated patients with at least 10000 HCV copies/ml were analyzed by NGS.
HCV amplicons (20 µL) were purified using 36 µL Agencourt AMPure XP beads (Beckman Coulter Inc). Amplicon concentrations were determined with the Invitrogen™ Quant-iT™ PicoGreen® dsDNA Assay (Invitrogen, Inc.). Purified amplicons were diluted to 5.0 ng/µl and 2 µL were processed using the Illumina Nextera XT DNA Library Preparation Kit (Illumina Inc.) and uniquely indexed using the Illumina Nextera XT Index Kit (96 Index). All amplified Nextera-PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter Inc.). All recovered DNA was pooled in equal volumes at a normalized concentration (1.43 ng/µl, the lowest concentration recovered). The DNA concentration of the pool was determined using the Invitrogen™ Quant-iT™ PicoGreen® dsDNA Assay (Invitrogen,Inc.). Library quality was determined using a Bioanalyzer 2100 (Agilent Technologies) and was sequenced on a MiSeq deep sequencing platform (Illumina, San Diego, CA) using a the 500-cycle MiSeq reagent kit v2.Fastq files were generated using the onboard MiSeq Reporter.

PCR amplicons were cleaned up before adaptor addition using the Ampure XP magnetic bead system (Beckmann Coulter, MA, United States) according to manufacturer’s recommendation. Dual barcode indices and sequencing adaptors were attached to each amplicon using the Illumina Nextera XT Index kit (Illumina, Inc., San Diego, CA, United States) following the manufacturer’s protocol, followed by a further Ampure XP cleanup step. Purified amplicons were quantified using the Quant-iT^TM PicoGreen^TM dsDNA Assay Kit (Thermo Fisher Scientific) and a Tecan Safire microplate reader (Tecan Group, Männedorf, Switzerland). Equimolecular amounts from each individual sample in 10 mM of Tris were combined, and the pooled library was additionally purified with two rounds of Ampure XP cleanup step. The library was sequenced by the Genomics Unit at “Fundación Parque Científico de Madrid” (Madrid, Spain) using the Illumina MiSeq platform (Nano-V2; PE 2x 250 bp). The ZymoBIOMICS microbial standard (Zymo Research Corp., Irvine, CA, United States) and water (no template DNA) were used as internal positive and negative controls, respectively, for library construction and sequencing. Raw sequence data have been deposited in the Sequence Read Archive (SRA) database at the NCBI under BioProject accession number PRJNA684121.

Genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue protocol (Qiagen, Valencia, CA) for Gram-negative bacteria and eluted with ultrapure water. Extracted DNA was quantified using a Qiagen QIAxpert system and Picogreen assay. The presence of Mycoplasma spp. was verified with qPCR [21 ], with the assumption that either or both of M. agassizii and M. testudineum may have grown from the tortoise sample cultures. Genomic sequencing was performed using the Illumina Nextera XT DNA Library Preparation Kit (Illumina, Inc., San Diego, USA) with the Illumina NextSeq500 platform (150bp, paired-end) and up to 2 ng of DNA per sample at the Nevada Genomics Center (University of Nevada, Reno). Sequencing was achieved in a multiplex system, using dual index sequences from the Illumina Nextera XT Index kit (index 1, N701 to N715; index 2 S502 to S511). Raw sequences were deposited in the NCBI Sequence Read Archive (SRA) database under bioproject PRJNA655797.

Analysis of the 16S rDNA of the microbial community present in feces was performed in accordance with a method described previously^{18 (link)} with minor modifications. In brief, the V3-V4 region of 16S rDNA was amplified using the primers as previously reported^{18 (link)}, and then ligated with overhang Illumina adapter consensus sequences. After PCR reactions,the amplicon was purified using AMPure XP magnetic beads (Beckman Coulter, Brea CA, USA). The Illumina Nextera XT Index kit (Illumina) with dual 8-base indices was used to allow for multiplexing. To incorporate two unique indices to the 16S amplicons, PCR reactions were performed as previously described^{19 (link)}. The libraries were purified by AMPure XP beads, quantified fluorometrically using a QuantiT PicoGreen ds DNA Assay Kit (Invitrogen, Paisley, UK) and then diluted to 4 nM using 10 mM Tris-HCl (pH 8.0), followed by pooling of the same volume for multiplex sequencing. The multiplexed library pool (10 pM) was spiked with 40% PhiX control DNA (10 pM) to improve base calling during sequencing. Sequencing was conducted using a 2 × 250-bp paired-end run on a MiSeq platform with MiSeq Reagent Kit v2 chemistry (Illumina).