Truseq nano dna library prep kit

DNA from samples 3-4D, 5-3D, 6-6B, 5-2D, 5-4B, 2-2B, 2-3B, 2-4A, 3-3A and 3-4A was prepared with TruSeq Nano DNA Library Prep Kit (Illumina) according to the manufacturer's protocol: 100 ng of genomic DNA were fragmented to 350 bp using Covaris LE220 system (Covaris, Inc.). Fragments were end-repaired, A-tailed, adaptor ligated and PCR amplified (8 cycles). The final libraries were validated using Agilent Tapestation 2200 (Agilent Technologies) and Qubit flourometer (Invitrogen), normalized and pooled in equimolar ratios. 101 bp paired-end sequencing was performed on the Illumina HiSeq 4000 according to the manufacturer's protocol. To match downstream analysis requirements, only the first members of the read pairs were considered, and were truncated to the first 50 bp. The resulting genome coverage was between 0.25 and 0.39 × (0.64–0.79 × for paired end reads). Data analysis were performed following Scheinin et al.49 (link)

Whole-genome sequencing (WGS) was performed for 25 trios of Lithuanian origin at coverage of 26.88–61.38× (an average of 36.27×), Table S1. Overall, the sample size was 75 individuals: 25 newborns, 25 mothers, and 25 fathers. WGS was performed at the CeGaT company (Tubingen, Germany). 100 ng DNA was paired-end sequenced in 2 × 150 bp mode on state-of-the-art Illumina NovaSeq™ 6000 Sequencing System using TruSeq^® Nano DNA Library Prep Kit (Illumina Inc., San Diego, CA, USA).
Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq (2.20). Adapters were trimmed with Skewer (version 0.2.2) [10 (link)]. Quality trimming of the reads has not been performed. Analysis of sequencing data was performed using the Illumina DRAGEN platform (version 3.6.4). The DRAGEN DNA Pipeline uses the current industry standard, BWA-MEM and GATK-HC software. Reads were mapped to the reference genome hg19 (present on the Illumina DRAGEN platform v.3.6.4) and duplicates were marked. Calling of small variants, regions of homozygosity, and structural variants was performed with default parameters. SNVs found at higher frequencies than 1% in the population were qualified as SNPs. The quality of the FASTQ files was analyzed with FastQC (version 0.11.5-cegat) [11 ]. Sequencing quality control Q30 values were above 88.59% (Figures S1–S3).

For analysis of LOH in ESCs and allelic dropouts in SCNT-embryos and donor fibroblasts, informative genomic variants were uncovered in gametes and fibroblast donors using whole-genome sequencing (WGS). WGS libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina) following the manufacturer’s instructions. Paired-end sequencing was performed on Illumina NovaSeq 6000 platform as 2 × 151 bp at an average coverage depth of 77.46X and uniformity of 96.13. Raw fastq files were uploaded to the Illumina BaseSpace Sequencing Hub for downstream processing. Genomic reads were aligned against GRCh38 human genome assembly and SNVs and indels were called using Dragen Germline Pipeline Version 3.6.3. For easier identification of informative heterozygous genomic variants and phasing, respective trio gVCFs were further subjected to Dragen Joint Genotyping Pipeline 3.6.3.

The genomic DNA was extracted and purified from RD012353 cells grown in 15 ml of MRS medium containing d-fructose with a MasterPure Gram Positive DNA Purification Kit (epicenter, WI, USA). A library was constructed using the TruSeq Nano DNA Library Prep Kit (Illumina, CA, USA), and the library was sequenced using the Illumina MiSeq platform (301-bp paired-end). The raw reads were cleaned up using Trimmomatic ver. 0.36^{35 (link)} by trimming adapter sequences and low-quality reads with the following parameters: CROP:300, SLIDINGWINDOW:4:15, and MINLEN:150. The resultant 628,975 high-quality read pairs totaling 307.3 Mb were assembled using SPAdes ver. 3.13.0^{36 (link)} with a default set of k-mer sizes and options “-careful and -cov-cutoff auto”. The draft genome sequence of RD012353 was annotated using DFAST-core ver. 1.2.6^{37 (link)} with an in-house database created from 10 genome information of the genus Fructobacillus deposited in the NCBI RefSeq database (July 1, 2020), and the annotations were manually curated. The predicted gene products were also annotated using KofamKOALA ver. 2020-06-07^{38 (link)} and InterProScan ver. 5.45-80.0^{26 (link)} to find candidate enzymes involved in the NMN biosynthetic pathway.

Amplified dsDNA was subjected to library preparation using TruSeq Nano DNA library prep kit (Illumina). Samples were subjected to end-repair during 30 min at 30°C. Size selection was performed using Agencourt AMPure XP beads in two steps: removal of large DNA fragments (>450 bp) with a ratio 1.6:1 of diluted beads:sample; removal of small DNA fragments (<150 bp) with a ratio 1:3.1 of undiluted beads:sample. DNA of interest was recovered in TE; 3’ ends of 150–450 bp DNA fragments were adenylated using A-Tailing Mix during 30 min at 37°C followed by enzyme deactivation. Then, Illumina adapters were ligated using Ligation Mix 2, Resuspension Buffer and the appropriate barcoded adapter in each case. As a last step, two sets of clean ups were performed using AMPure XP beads: first in a 1.2:1 ratio and next in a 1.1:1 ratio. DNA was eluted with TE and PCR amplified using PCR Primer Cocktail and Enhanced PCR Mix as follows: 3 min at 95°C, and 8 cycles of 20 s at 98°C, 15 s at 60°C and 30 s at 70°C. As a final step, the final amplified libraries were cleaned up using Sample Purification Beads. The size of the library was determined by HS-DNA Agilent Bioanalyzer (150–450 bp). The amount of library was determined by Nanodrop and dsDNA Quantifluor. Quality checks were performed comparing with aliquots taken from the >450 bp and <150 bp fractions.