Genomic dna screentape assay

Before genome sequencing, the quality/quantity of DNA samples were assessed using the Agilent Genomic DNA ScreenTape assay in conjunction with the 4200 TapeStation system (Agilent Technologies). The 10 ug of total DNA was sonicated using a Covaris M220 Focused-ultrasonicator to a size ranging from 400 to 500 bp. Subsequently, genomic DNA was used for library preparation using the Illumina Truseq DNA Sample Preparation Kit (Illumina, San Diego, USA), following the manufacturers’ preparation protocol. Genome sequencing was performed using the Illumina HiSeq platform for PE 2 × 150 bp sequencing. The raw sequences were filtered to obtain qualified reads using FASTP v.0.20 (Chen et al. 2018 (link)) and FLASH v.1.2 was used to merge paired-end reads (Magoč and Salzberg 2011 (link)). The complete circular mitochondrial genome of M.p.pentadactyla was assembled de novo with MitoFinder v.1.3 (Allio et al. 2020 (link)). Approximately 32.34 GB of clean data were obtained, yielding about 2192-fold depth of coverage of the mitogenome.

Genomic DNA from the 22 environmental strains tested on HeLa cells was isolated as described above. The quantity was measured using both a Qubit 2.0 fluorometer with a Qubit dsDNA HS assay kit (Invitrogen, Thermo Fisher Scientific) and a 2200 TapeStation instrument with a genomic DNA ScreenTape assay (Agilent Technologies Inc., Santa Clara, CA, USA). Genomic libraries were prepared using a Nextera XT kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol and quantified by capillary electrophoresis, applying the Agilent High Sensitivity D5000 ScreenTape system (Agilent Technologies Inc.). Libraries were sequenced on a MiSeq machine (Illumina) using v2 reagents with 2 × 250-bp paired-end reads. Consequently, 81.6% to 98.8% of bases in sequencing reads had quality scores of 30 (Q30) or higher. De novo genome assembly was performed using CLC Genomic Workbench v5 (Qiagen GmbH). Rapid Annotation using Subsystem Technology (RAST server; https://rast.nmpdr.org/) was used for functional annotation of proteins (61 (link)).

Genomic DNA was isolated from refrozen blood samples by Invisorb QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) or Spin Blood Mini Kit (Stratec Molecular, Birkenfeld, Germany) according to the manufacturer’s instructions. The quality and quantity of the DNA samples were determined using DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA) and Qubit 3.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) using Qubit dsDNA HS Assay (ThermoFisher Scientific, Waltham, MA, USA). Integrity of DNA was determined by capillary electrophoresis on TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) using Genomic DNA ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA).

For the discovery group and second validation dataset, DNA was extracted from whole blood using the QIAamp DNA Blood Mini Kit™ (Qiagen, Venlo, The Netherlands,). Extracted DNA was quantified using the Qbit (Invitrogen, Waltham, MA, USA) and DNA integrity was assessed using the Genomic DNA ScreenTape assay with the TapeStation system (Agilent, Santa Clara, CA, USA), using the DNA integrity number (DIN) as a metric. All samples had a DIN ≥ 7, indicating minimal genomic DNA degradation.

DNA integrity number was estimated using a Genomic DNA Screen Tape Assay (Agilent). The PReCR mix (NEB) was used after fragmentation of the DNA (Covaris E220: Duty cycle = 10%, Peak Intensity = 175, Cycles = 200, Time = 240 s) to overcome amplification bias due to DNA damage during the library preparation and to improve the single nucleotide polymorphism calling efficiency. After bead clean up (AMPure beads, Beckman Coulter) Illumina’s TruSeq DNA Nano library kit was used with slight modifications: no size selection, 13 cycles of PCR. The samples were enriched for the whole exome with Agilent’s Sure Select Target enrichment regents (V6 + UTR). To block the complete P5/P7 adapter structure of the library molecules, the IDT blocking reagent for TruSeq DNA Nano libraries replaced the blocking oligos of the Agilent protocol. Accordingly, the PCR primers used during post-capture PCR were the TruSeq DNA Nano Primer Cocktail Mix (Illumina), and the annealing temperature was set to 60 °C. The exome-enriched libraries were sequenced on Illumina’s HiSeq 4000 in 75-bp paired-end mode.

Genomic DNA was isolated from biopsy tissue sections using a standard protocol. In particular, paraffin was removed from formalin-fixed paraffin-embedded (FFPE) samples with Bio-Clear (Bio-optica, Milan, Italy), and DNA was purified using the QIAamp DNA FFPE Tissue kit (Qiagen, Valencia, CA, USA). DNA quantitation and quality assessment were carried out with both a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and Qubit^® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). DNA fragmentation status was evaluated with an Agilent 2200 TapeStation system using Genomic DNA ScreenTape assay (Agilent Technologies, Santa Clara, CA, USA) able to produce a DNA integrity number (DIN). Quantitative measurements of EGFR mutations were performed using the Therascreen™ EGFR Pyro Kit (Qiagen), for the detection of mutations in codon 719 (exon 18), in codons 768 and 790 (exon 20), and in codons 858 to 861 (exon 21), along with deletions and complex mutations in exon 19 of the human EGFR gene on genomic DNA. Each pyrosequencing assay was performed on the PyroMark Q24 system (Qiagen), following the manufacturer’s instructions.

Quality and quantity of DNA was measured using the Genomic DNA Screen Tape Assay (Agilent Technologies, Santa Clara, CA) and Qubit. The concentration of genomic DNA (gDNA) larger than 200 bp was then calculated, and at least 200 ng of DNA greater than 200 bp was sheared in 50 µL of nuclease-free water with the Covaris E220 using the 96 microTUBE Plate (Covaris, Woburn, MA). The library was prepared using KAPA Hyper Prep Kit (Roche, Basel, Switzerland) and 100–500 ng of sheared DNA according to the manufacturer’s instructions. Individual tumor adapter-ligated libraries were enriched into the exome capture reaction, and for germline each adapter-ligated library was pooled before proceeding to capture using Agilent’s SureSelect Human All Exon V6 + custom probes capture library kit [28 (link)]. Samples that had successful libraries created were then sequenced on Illumina MiSeq technology for quality control to assess the ability of the libraries to be sequenced. Subsequently, each library was pooled and sequenced on Illumina’s NovaSeq 6000 (Illumina, San Diego, CA) using 300 cycle kit. Raw FASTQs were generated using the industry standard BCL2FASTQ v1.8.4