Genomic dna reagents

The quantity and concentration of DNA present was measured in triplicate using a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, Q32853) and a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Q33216). Absorbance values (to measure contamination) were taken using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Size measurements were taken with either a Tapestation 2200 (Agilent Technologies, Santa Clara, CA, USA) and Genomic DNA Screentape (Agilent Technologies, 5067-5365) and Genomic DNA Reagents (Agilent Technologies, 5067-5366), or a Femto Pulse (Agilent Technologies) and the Genomic DNA 165 kb Kit (Agilent Technologies, FP-1002-0275). To size-evaluate the high molecular weight DNA, GQN (Genomic quality number) analyses of Femto Pulse runs were carried out using Prosize (Agilent Technologies) in gDNA mode; for example, a GQN_50kb of 0.5 indicates that 50% of fragments are longer than 50 kb in the total DNA [19 ].

Three samples of each genome containing approximately 4 × 10¹¹ copies of AAV vector genome were aliquoted. Two of the samples were treated with DNase I (New England Biolabs) at 37°C. After 60 min, 5 mM EDTA (Sigma) was added to both samples to quench the reaction. All three samples (DNase I treated and untreated) were then heated to 80°C for 10 min. Conversion of ssDNA to dsDNA was performed by transferring the samples to a water bath at 65°C, and then the water bath was turned off so that the temperature dropped to 40°C–43°C during the course of 60 min. The DNA in one of the samples treated with DNase I was purified using a QIAquick PCR purification kit (QIAGEN). 2-μL samples of vector DNA and 10 μL of sample buffer (Agilent) were vortexed at 2,200 rpm for 1 min. Genomic DNA ScreenTape (Agilent, PN 5067-5365) and genomic DNA reagents (Agilent, PN 5067-5366) were used to measure the size of the dsDNA using an Agilent 4200 TapeStation.

To minimize infections and laboratory contamination sterile surgical gloves and face masks (for collecting samples) were used and all DNA extraction steps were performed with sterile or sterilized equipments in a class II laminar air-flow cabinet. Negative isolation control (NIC) experiments were simultaneously conducted by substituting samples with PCR grade water. Elutes of the NIC samples were conveyed for V3-V4 amplicon - PCR and indexing performed under DNA free UV sterilized AirClean® PCR workstations/cabinets. At each stage the PCR clean-ups of the library preparation NIC amplicons were also validated on 4200 Tape Station System (G2991AA, Agilent Technologies; Santa Clara, California, United States) by Agilent D1000 ScreenTapes (5067–5365) and Agilent Genomic DNA reagents. Host background nucleic acid contaminations were also detected by conducting real-time PCR reactions using GAPDH assay on eluted gDNAs. For measuring the overall quality of Illumina MiSeq paired-end (PE, 2 × 301 nt) sequencing runs 5% PhiX spike-in quality control (PhiX Control Kit v3 - FC-110-3001) was used.

Genomic DNA for long-read sequencing was isolated using the Wizard HMW DNA Extraction Kit (Promega) following the manufacturer’s instructions. The obtained gDNA was quantified using the Qubit fluorometer with the Qubit dsDNA BR-Assay-Kit (Thermo Scientific). DNA quality and purity were determined using NanoDrop 2000 (Thermo Scientific) absorbance measurements at 230, 260 and 280 nm. Integrity and fragment length distribution of obtained gDNA were analysed with the 4150 TapeStation System using Genomic DNA reagents and ScreenTape (Agilent Technologies).