Qiaxcel dna high resolution kit

The QIAseq Targeted DNA technology from Qiagen was used to develop kits for our customized gene panel composed of the selected 96 genes (Cat. No. EDHS-10082-002Z-3002 and CDHS-12403Z-675 Qiagen, Hilden, NRW, Germany). The NGS libraries were constructed according to the manufacturer’s instructions. After library preparation, the QIAxcel (Cat No. 900194 Qiagen, Hilden, NRW, Germany) was used to check the fragment size and concentration with the QIAxcel DNA high resolution kit (Cat No. 929002, Qiagen, Hilden, NRW, Germany). The prepared libraries were quantified using QIAseq Library Quant Assay Kit (Cat No. 333304, Qiagen, Hilden, NRW, Germany). Then, the libraries with different sample indexes were combined in equimolar amounts to achieve a similar sequencing depth for each combined library. The fragment size distribution in our libraries ranged between 200 and 1000 bp. The Ion PI Hi-Q Chef Kit (Cat. No. A27198, Thermo Fischer Scientific Inc., Waltham, MA, USA), running on the Ion Chef, was used to load the combined libraries on the Ion PI Chip (Cat. No. A26770, Thermo Fischer Scientific Inc., Waltham, MA, USA). The Ion Proton Platform was used for next generation sequencing using the Ion Proton Sequencing 200 Kit v2 (Cat. No. 4485149, Thermo Fischer Scientific Inc., Waltham, MA, USA).

Previously identified anthracnose resistance molecular markers, polymorphic between Nautica and B09197, were used to genotype the RIL population. The SCAR markers used with linkage to Co–4 were SAS13 [38 (link)], SH18 and SBB14 [47 ]. PCR amplifications were performed in 25 μL reaction volumes with 1 μL genomic DNA (25 ng), 0.4 units of GOTaq DNA polymerase (Promega), 2.5 μL 10 X polymerase buffer, 200 mM dNTP, and 1 mM MgCl₂ and 0.15mM each primer. Amplification conditions were 3 min at 94°C followed by 35 cycles of 10 s at 94°C, 30 s at 60°C and 1 min at 72°C. The PCR amplifications were performed in a Master Cycler nexus GSx1 (Eppendorf) using clear unskirted 96-well MultiplateTM PCR plate (BIO-RAD). The PCR products were separated with the Qiaxcel advanced capillary (QIAGEN) system using either QIAxcel DNA High Resolution Kit or QIAxcel DNA Fast Analysis Kit according to the manufacturer’s protocol.

We employed the QIAseq Human BRCA1, and BRCA2 targeted DNA panel (Qiagen, Germany, Cat. No. DHS-102Z). The manufacturer's instructions were followed while building the NGS libraries. Then, the fragment size and concentration were assessed using the QIAxcel DNA high-resolution kit (Cat No. 929002, Qiagen, Hilden, NRW, Germany). The libraries were subsequently quantified using the QIAseq Library Quant Assay Kit (Cat No. 333304, available from Qiagen, Hilden, NRW, Germany). The template was prepared using the Ion PI Hi-Q Chef Kit (Cat. No. A27198, Thermo Fischer Scientific Inc., USA), and sequencing on the Ion Proton Platform was done using the Ion Proton Sequencing 200 Kit v2 (Cat. No. 4485149, Thermo Fischer Scientific Inc., USA).

We used QIAseq targeted DNA panel (human breast cancer panel) (Cat. No. 333505, Qiagen, Germany). This panel included 93 BC specific genes (ACVR1B, AKT1, APC, AR, ATM, ATR, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP, CASP8, CBFB, CCND1, CDH1, CDK4, CDK6, CDKN2A, CHEK2, CSMD1, CTNNB1, DIRAS3, EGFR, EP300, EPCAM, ERBB2, ERBB3, ERCC4, ESR1, EXOC2, EXT2, FAM175A, FANCC, FBXO32, FGFR1, FGFR2, GATA3, GEN1, HERC1, HOXB13, IRAK4, ITCH, KMT2C, KRAS, MAP2K4, MAP3K1, MDM2, MED12, MEN1, MLH1, MRE11A, MSH2, MSH6, MUC16, MUTYH, MYC, NBN, NCOR1, NEK2, NF1, PALB2, PALLD, PBRM1, PCGF2, PIK3CA, PIK3R1, PMS1, PMS2, PPM1L, PTEN, PTGFR, RAD50, RAD51, RAD51C, RAD51D, RB1, RET, SEPT9, SMAD4, SMARCA4, STK11, SYNE1, TGFB1, TP53, TRAF5, VHL, WEE1, XRCC2, XRCC3, and ZBED4).
The NGS libraries were constructed according to the manufacturer’s instructions. Then, the fragment size and concentration were checked using QIAxcel DNA high-resolution kit (Cat No. 929002, Qiagen, Hilden, NRW, Germany). Then, libraries were quantified using QIAseq Library Quant Assay Kit (Cat No. 333304, Qiagen, Hilden, NRW, Germany). The Ion PI Hi-Q Chef Kit (Cat. No. A27198, Thermo Fischer Scientific Inc., Waltham, MA, USA) was used for template preparation and the Ion Proton Sequencing 200 Kit v2 (Cat. No. 4485149, Thermo Fischer Scientific Inc., Waltham, MA, USA) was used for sequencing on the Ion Proton Platform.

The viral DNA was extracted from infected cells by using a QIAamp DNA mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. For the typing of HAdV, the penton base, hexon, and fiber gene sequences were obtained for all HAdV strains; PCR was performed with the Platinum PCR SuperMix (Invitrogen) following the manufacturer instructions. The primer pairs designed to amplify and sequence the penton base, hexon, and fiber gene sequences are listed in Table 1. The amplification products were analysed with capillary gel electrophoresis by using the QIAxcel DNA High Resolution Kit (Qiagen, the Netherlands). After the PCR products were purified with a QIA Gel Extraction Kit (Qiagen, Valencia, CA, USA), the amplicons were bi-directionally sequenced using the Sanger sequencing method (BigDye Terminator, Version 3.1, Cycle Sequencing kit; Life Technologies, Grand Island, NY, USA) and an ABI PRISM 3130 genetic analyser (Applied Biosystems, Foster City, CA, USA).

The PCR method with specific oligonucleotide primers (Table S1) was performed according to Fries et al. (2013) [30 ]. Visualization of amplified PCR products was performed using the QIAxcel electrophoresis system with a QIAxcel DNA high-resolution kit (Qiagen, Hilden, Germany). The analysis was processed using the QIAxcel Screen Gel software system 1.5.0. The size of the segments of each product was determined by comparison with the size marker (50 bp–800 bp, Qiagen, Hilden, Germany) and alignment marker (15 bp–1 kbp, Qiagen).