The genetic diversity analysis of I. cangae individuals was performed using the genome skimming approach (Figure 2a ) (Malé et al., 2014 (link); Weitemier et al., 2014 (link); Wessinger et al., 2018 (link)). Shotgun paired‐end libraries were constructed from 50 ng of isolated DNA. For that, samples were subjected to a random enzymatic fragmentation in which the DNA was simultaneously fragmented and bound to adapters using the QXT SureSelect kit (Agilent Technologies). The fragmented DNA was purified using AmPure XP beads (Beckman Coulter) and subjected to an amplification reaction using primers complementary to the Illumina flowcell adapters. Amplified libraries were again purified using AmPure XP beads (Beckman Coulter), quantified using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc.), and checked for fragments size in the 4,200 TapeStation (Agilent Technologies®) using a ScreenTape DNA 1,000 kit (Agilent Technologies). The libraries were adjusted to a 4 nM concentration, pooled, denatured, and diluted to a running concentration of 1.8 pM. The sequencing run was performed in the NextSeq 500 Illumina platform using a NextSeq 500 v2 kit high output (300 cycles).
Sureselect qxt kit
Manufactured by Agilent Technologies
Sourced in United States
The SureSelect QXT kit is a library preparation solution for next-generation sequencing (NGS) applications. It is designed to streamline the library preparation process by automating several steps, reducing hands-on time and increasing efficiency. The kit includes reagents and consumables necessary for DNA fragmentation, end-repair, A-tailing, and adapter ligation, enabling the generation of sequencing-ready libraries from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Qiasymphony, by Qiagen (2 mentions)
Autopure, by Qiagen (2 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Miseq platform, by Illumina (2 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 v2 kit, by Illumina (1 mentions)
Qubit 3, by Thermo Fisher Scientific (1 mentions)
Suredesign, by Agilent Technologies (1 mentions)
High sensitivity dna kit, by Agilent Technologies (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Targetseq exome enrichment kit, by Thermo Fisher Scientific (1 mentions)
Solid 5500 xl sequencing platform, by Thermo Fisher Scientific (1 mentions)
Miseq v3 600 cycles kit, by Illumina (1 mentions)
10 protocols using sureselect qxt kit
1
Genome Skimming for Genetic Diversity Analysis
2
Plant DNA Extraction and Sequencing
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DNA extractions were carried out following the automated protocol previously described [51 (link)], with approximately 20 mg of plant material collected in NaCl-saturated CTAB solution [52 ]. Afterwards, paired-end libraries were constructed from 50 ng of DNA. Samples were subjected to a step of enzymatic and random fragmentation in which the DNA were simultaneously fragmented and bound to adapters using the QXT SureSelect kit (Agilent Technologies) according to the manufacturer’s instructions. The fragmented DNA was purified and subjected to an amplification reaction using primers complementary to the adapters. Next, the libraries were quantified using the Qubit 3.0 (Invitrogen) fluorimeter and checked for fragments size in the 2100 Bioanalyzer (Agilent Technologies). Then, the libraries were diluted in a solution of 0.1% Tris-HCl and Tween and pooled. The sequencing run was performed with a NextSeq 500 v2 kit high-output (300 cycles).
3
Genetic Profiling of ALS Genes
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DNA was extracted from peripheral blood. Next‐generation sequencing analysis was performed by means of amplicon deep sequencing using Sure Select QXT kit (Agilent) for ALS causative genes (SOD1, FUS, TARDBP, VCP, OPTN, SQSTM1, TUBA4A, PFN1, and UBQLN2) and repeat primed PCR AmplideX PCR/CE C9ORF72 kit (Asuragen Inc., Austin TX) for detection of C9orf72 expansion.
4
Exome Sequencing of Sorted Mouse Tumor Cells
5
Comprehensive mtDNA Genome Sequencing
6
Genetic Diagnostic Analysis of Pulmonary Arterial Hypertension
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DNA was extracted from 3–10 mL of EDTA-blood samples using an automated procedure (Autopure or QIAsymphony, Qiagen, Hilden, Germany). Genetic diagnostic analysis was carried out with our patented (EP3507380) PAH-specific gene panel, including the following PAH diagnostic genes: ACVRL1, AQP1, ATP13A3, BMPR1B, BMPR2, CAV1, EIF2AK4, ENG, GDF2, KCNA5, KCNK3, KLF2, SMAD4, SMAD9, SOX17 and TBX4 (customized SureSelect QXT kit, Agilent, Germany). The procedure was carried out as previously described [2 (link),11 (link)].
Multiplex ligation-dependent probe amplification (MLPA) was performed in addition to identify exon deletions and duplications in the genes ACVRL1, BMPR2 and ENG (P093-C2, MRC-Holland, Amsterdam, The Netherlands). Familial variants were sought by Sanger sequencing (ABI Genetic Analyzer 3130xl, Applied Biosystems, MA, USA) or MLPA. Variants were characterized following the American College of Medical Genetics and Genomics guidelines [12 (link)].
Multiplex ligation-dependent probe amplification (MLPA) was performed in addition to identify exon deletions and duplications in the genes ACVRL1, BMPR2 and ENG (P093-C2, MRC-Holland, Amsterdam, The Netherlands). Familial variants were sought by Sanger sequencing (ABI Genetic Analyzer 3130xl, Applied Biosystems, MA, USA) or MLPA. Variants were characterized following the American College of Medical Genetics and Genomics guidelines [12 (link)].
7
Targeted Hydrocephalus Gene Sequencing
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A total of 11 genes were targeted for capture and deep sequencing. They include the 3 validated genes involved in non-syndromic hydrocephalus, L1CAM, MPDZ and CCDC88C, as well as eight candidate genes on the basis of mouse models of hydrocephalus (panel list available upon request). Using Sure Design (Agilent Technologies Inc., Santa Clara, California, USA), the capture was designed to include the whole L1CAM gene and all the coding exons from the 10 other genes with at least 50 bp of the flanking intronic sequence. In addition, 150 pb of the 5’UTR and 3’UTR regions were added to the targeted regions. NGS library preparations and target enrichment were performed with the SureSelect QXT kit (Agilent Technologies Inc., Santa Clara, California, USA): in-solution hybridization capture using custom biotinylated RNA baits was performed according to the manufacturer’s instructions. The multiplexed samples were sequenced on the Illumina Miseq platform (Illumina, San Diego, California, USA) using 150-bp paired-end reads.
8
Exome Sequencing for Mutation Analysis
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To analyse the mutation profile of clones, exome sequencing was performed after UVA irradiation. Libraries were prepared with 5 μg of genomic DNA using the Sure Select QXT kit (Agilent Technologies, Santa Clara, CA, USA). Sure Select QXT Human All Exon V6 baits were used for the exome capture procedure. Library preparation and the exon capture were carried out following the manufacturer's specifications. Quality control of libraries was performed with two kits: a High Sensitivity DNA kit (Agilent Technologies) and an Illumina library Quantification kit with primer premix and Light Cycler 480 qPCR mix (KAPA Biosystems, Wilmington, MA, USA). Libraries were sequenced in paired-end mode (2 × 125 bp) on the Illumina HiSeq platform (Illumina, San Diego, CA, USA). Similar but entirely independent experiments were performed using the SOLiD 5500xl sequencing platform (Applied Biosystems, Cleveland, Ohio, EUA) in paired-end mode (75 × 35 bp) with the TargetSeq Exome Enrichment Kit (Life Technologies, Carlsbad, CA, USA). The results are shown in detail in the Supplementary data (Supplementary Figures S2 and S3 ).
9
DNA Extraction and Sequencing of Serjania erecta
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We collected leaves from an individual of Serjania erecta in the Ecocerrado Brasil Private Heritage Reserve in Araxá, Minas Gerais, Brazil (19°36’47.1” S 47°08’20.9” W altitude 939 m) for DNA extraction. The species was determined using the botanical identification key for the genus (Somner et al., 2015 ). Plant material was identified by Dr. Inês Cordeiro (Instituto Botânico, São Paulo, São Paulo, Brazil), and a voucher specimen was deposited in the Herbarium of Medicinal Plants at UNAERP with voucher number HPMU-835.
Total DNA was extracted using the CTAB protocol (Doyle and Doyle, 1987 ) and quantified using horizontal agarose gel electrophoresis (1%). The library for sequencing was constructed using the SureSelectQXT kit (catalog number 5500-0120, Agilent Technologies), and the library quality validation was performed using the Bioanalyzer 2100 (Agilent). Subsequently, the library was sequenced on the MiSeq platform (Illumina) in paired-end mode (2x300) using the MiSeq v3 600 cycles kit (Illumina). All the wet laboratory steps were conducted at the Laboratory of Genetics & Biodiversity – LGBio, at the Federal University of Goiás, in Goiânia (GO) – Brazil.
Total DNA was extracted using the CTAB protocol (Doyle and Doyle, 1987 ) and quantified using horizontal agarose gel electrophoresis (1%). The library for sequencing was constructed using the SureSelectQXT kit (catalog number 5500-0120, Agilent Technologies), and the library quality validation was performed using the Bioanalyzer 2100 (Agilent). Subsequently, the library was sequenced on the MiSeq platform (Illumina) in paired-end mode (2x300) using the MiSeq v3 600 cycles kit (Illumina). All the wet laboratory steps were conducted at the Laboratory of Genetics & Biodiversity – LGBio, at the Federal University of Goiás, in Goiânia (GO) – Brazil.
10
Genetic Analysis of Pulmonary Arterial Hypertension
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EDTA‐blood samples were used to extract DNA by an automated procedure (Autopure or QIAsymphony; Qiagen). Next generation sequencing data was obtained with a patented (EP3507380) PAH‐specific gene panel including all currently known PAH genes (customized SureSelect QXT kit; Agilent) as described previously.2 Multiplex ligation‐dependent probe amplification (MLPA) was used to identify exon deletions and duplications in the genes ACVRL1, BMPR2, and ENG (P093‐C2; MRC‐Holland). Familial variants were sought by Sanger sequencing (ABI Genetic Analyzer 3130xl; Applied Biosystems) or MLPA. Variants were classified following the American College of Medical Genetics and Genomics guidelines.17 Disease‐causing pathogenic variants and likely pathogenic variants were defined for this work as “mutations.”
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