sureselectxt kit, by Agilent Technologies - Product details

1

HAdV DNA Library Preparation and Sequencing

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Library preparation of 42 specimens (including all cell culture isolates) with sufficient HAdV DNA load (a ratio of HAdV/human genomic DNA >1,000) was performed using Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) with 1 ng of DNA as input following manufacturers’ guideline. Purified DNA of the other nine specimens (6C1, 19C2, 20C1, 23C2, 24C2, 35C2, 47C2, 48C2 and 50C1) was sheared by sonication, and sequencing libraries were prepared directly (KAPA library preparation kit, KAPA Biosystems, Wilmington, MA, USA) as previously described^{49 (link)}, with a few modifications. DNA fragments were end-repaired, A-tailed and ligated to NEBnext Illumina adapter (New England Biolabs, Ipswich, MA, USA). After PCR pre-amplification (6–14 cycles) with adapter specific primers, up to 750 ng of DNA was target enriched for HAdV fragments with RNA baits. These 120-mer RNA baits were designed to span the length of the positive strand of 24 Genbank HAdV-C1, -C2 and -C5 reference genomes (SureSelectXT kit, Agilent Technologies, CA, USA).

Dhingra A., Hage E., Ganzenmueller T., Böttcher S., Hofmann J., Hamprecht K., Obermeier P., Rath B., Hausmann F., Dobner T, & Heim A. (2019). Molecular Evolution of Human Adenovirus (HAdV) Species C. Scientific Reports, 9, 1039.

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2

Whole Exome Sequencing of Mouse Tumors

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Whole exome sequencing (exome-seq) of mouse tumors was carried out using
tissue frozen in RNA later (Ambion) at -80 °C. DNA was isolated using
the DNeasy Blood & Tissue Kit (Qiagen). Exome capture was carried out
with 2ug of DNA using the Agilent SureSelectXT kit. Sequencing to generate
paired-end100bp reads were obtained using isolated whole exomes sequenced on the
Illumina HiSeq 2500 platform. Our exome-seq pipeline produced a mean coverage of
195X within coding regions. Sequencing reads were aligned to the mouse reference
genome sequence (mm9) using BWA. SAM to BAM conversion was carried out using
Picard tools, and local realignment around indels with base quality score
recalibration was performed using the Genome Analysis Toolkit (GATK). Single
nucleotide variants (SNVs) were called using (GATK). The annotation of
non-synonymous SNVs was performed using Annovar. Comparison to human BCC
mutational landscape was performed using previously published variants
^{47 (link)}.
Smo mutation was identified by calling variants on
unfiltered alignment files using Samtools and annotated using snpEff using the
GRCm38.71 genome.

Whitson R.J., Lee A., Urman N.M., Mirza A., Yao C.Y., Brown A.S., Li J.R., Shankar G., Fry M.A., Atwood S.X., Hollmig S.T., Aasi S.Z., Sarin K.Y., Epstein EH J.r., Tang J.Y, & Oro A.E. (2018). Non-canonical hedgehog pathway activation by MKL1/SRF promotes drug-resistance in basal cell carcinomas. Nature medicine, 24(3), 271-281.

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3

Targeted Sequencing of M. tuberculosis

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A custom gene capture design was performed by Agilent design services (Agilent Technologies, USA), selecting 174 gene regions of the M. tuberculosis genome related to virulence and drug resistance (panel size 244 kbp).
According to Agilent’s Sure Select XT kit protocol, sequencing libraries were prepared using 200 ng of total DNA for each sample. Libraries quality was assessed with a Tape Station (Agilent), verifying fragment sizes of 225 to 275 base pairs (bp). Hybridization and capture were conducted with RNA probes, and targeted regions were purified with magnetic beads, followed by post-hybridization amplification for indexing. The NGS was performed using an Illumina NextSeq platform (2 × 150 bp) in pair-end reads.

Cuevas-Córdoba B., Fresno C., Haase-Hernández J.I., Barbosa-Amezcua M., Mata-Rocha M., Muñoz-Torrico M., Salazar-Lezama M.A., Martínez-Orozco J.A., Narváez-Díaz L.A., Salas-Hernández J., González-Covarrubias V, & Soberón X. (2021). A bioinformatics pipeline for Mycobacterium tuberculosis sequencing that cleans contaminant reads from sputum samples. PLoS ONE, 16(10), e0258774.

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4

NSCLC Tissue Sampling and DNA Extraction

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Microtome sections (5 µM) were prepared from FFPE-tissue of NSCLC samples and one object slide was HE stained for tumor evaluation by a pathologist. Tumor tissue was gained from the remaining slides by manual microdissection, or in case material was limited, enriched by Laser Capture Microdissection (Leica CTR6500). DNA extraction was performed either manually (Macharey Nagel) or semi-automated (Maxwell MDx, Promega). The library preparation for the samples was performed using the Agilent SureSelect XT Kit as per the manufacturers’ recommendations. Specimens were processed and stored at either Hematopathology Hamburg or at the Department of General Pathology and Pathological Anatomy, University Hospital Heidelberg.

Roeper J., Christopoulos P., Falk M., Heukamp L.C., Tiemann M., Stenzinger A., Thomas M, & Griesinger F. (2022). TP53 co-mutations as an independent prognostic factor in 2nd and further line therapy—EGFR mutated non-small cell lung cancer IV patients treated with osimertinib. Translational Lung Cancer Research, 11(1), 4-13.

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5

FFPE Tissue DNA Extraction and Exome Sequencing

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Genomic DNA was extracted from microdissected formalin-fixed paraffin-embedded (FFPE) tissue using a QIAamp DNA FFPE Tissue Kit (Qiagen). At least 250 ng of genomic DNA was used for each sample. DNA quality was assessed on a 4200 TapeStation (Agilent). Genomic DNA libraries were generated using the SureSelectXT Kit (Agilent Technologies), followed by exon enrichment using the SureSelectXT Human All Exon V7 bait set (Agilent Technologies). The resulting exon-enriched libraries were subjected to paired-end, 100-cycle sequencing performed on a HiSeq 4000 (Illumina). Sequencing results were analysed using an institutionally established pipeline for alignment and calling of single nucleotide variants (SNVs) and insertions or deletions (indels), which were annotated and ranked by putative pathogenicity using a workflow named “medal ceremony” and subsequently manually reviewed [18 (link)–20 (link)].

Chiang J., Li X., Jin H., Wu G., Lin T, & Ellison D.W. (2022). The molecular characteristics of low-grade and high-grade areas in desmoplastic infantile astrocytoma / ganglioglioma. Neuropathology and applied neurobiology, 48(4), e12801.

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6

Whole Exome Sequencing Library Preparation

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To prepare libraries for Whole Exome Sequencing, 100ng-1µg of genomic DNA was sheared with the Covaris S220 (Covaris, Woburn, MA, USA), followed by end-repair, 3’ end Adenylation and ligation with paired-end adaptors. Post ligation, 15µl of the purified libraries were PCR amplified, all the above steps were performed using the Agilent SureSelect^XT kit and every step was followed by DNA purification on a magnetic stand using AMPure XP Reagent beads (Beckman Coulter Genomics, Danvers, MA, USA). Afterward, size (approx 225-275 bp) and quantity (>800ng) were verified employing the Agilent Tapestation 2200 system followed by hybridization and probe capture using Exome SureSelect Human All Exon V6+UTR probes (Agilent). Dynabeads MyOne Streptavidin T1 magnetic beads (Thermofisher). Finally, Captured Libraries were amplified with 12 cycles of PCR using indexing primers containing 8-bp indices, followed by an amplification using AMPure XP beads (Beckman Coulter). Final libraries were checked for quality (each fragment size approx. 300-400 bp) and quantity using Agilent Tapestation 2200 system.

Desai S.S., K R.R., Jain A., Bawa P.S., Dutta P., Atre G., Subhash A., Rao V.U., J S., Srinivasan S, & Choudhary B. (2021). Multidimensional Mutational Profiling of the Indian HNSCC Sub-Population Provides IRAK1, a Novel Driver Gene and Potential Druggable Target. Frontiers in Oncology, 11, 723162.

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7

Targeted Sequencing of DCM Genes

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DNA was extracted using Qiagen’s Gentra Puregene kit. 1μg of DNA was sheared with a Diagenode BioRuptor for thirty minutes set on high with thirty seconds on/off. Sheared DNA underwent library prep using Agilent’s SureSelect XT kit. Barcoded libraries were enriched for DCM-associated genes using a custom Agilent SureSelect bait panel (Seidman DCMv5)^{7 (link)}, which includes all 364 exons of TTN. Libraries were run on an Illumina NextSeq and variants were called using Agilent’s SureCall.

Goli R., Li J., Brandimarto J., Levine L.D., Riis V., McAfee Q., DePalma S., Haghighi A., Seidman J.G., Seidman C.E., Jacoby D., Macones G., Judge D.P., Rana S., Margulies K.B., Cappola T.P., Alharethi R., Damp J., Hsich E., Elkayam U., Sheppard R., Alexis J.D., Boehmer J., Kamiya C., Gustafsson F., Damm P., Ersbøll A.S., Goland S., Hilfiker-Kleiner D., McNamara D.M, & Arany Z. (2021). Genetic and phenotypic landscape of peripartum cardiomyopathy. Circulation, 143(19), 1852-1862.

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8

Targeted X-Y Chromosome Sequencing

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A SureSelect targeted panel (Agilent Technologies, UK) was designed to capture the whole genomic region encompassing the telomeric 4 genes on the pseudoautosomal region of X and Y chromosomes, chrX:198061-607558 chrY:148061-557558 (hg19/GRCh37). Library preparation was by SureSelectXT kit under manufacturer’s instructions (Agilent Technologies, UK), and sequencing on NextSeq instrument 500/550, read length of 2 × 150 bp (Illumina, USA); Leeds melanoma samples (n = 168) and GOSH CMN samples (n = 5). BAM files were inputted to DeepTools MultiBamSummary using the PPP2R3B_Moderately_Stringent_1_covered.bed probe coordinates file. Coverage across probe regions within hg19 coordinates were extracted and averaged. Three control samples were used to create “normal expected” coverage ratios of the genes compared to SHOX. All samples were normalized compared to these ratios and R studio was used to visualize and calculate gene coverage data across all samples.

Polubothu S., Zecchin D., Al-Olabi L., Lionarons D.A., Harland M., Horswell S., Thomas A.C., Hunt L., Wlodarchak N., Aguilera P., Brand S., Bryant D., Carrera C., Chen H., Elgar G., Harwood C.A., Howell M., Larue L., Loughlin S., MacDonald J., Malvehy J., Barberan S.M., da Silva V.M., Molina M., Morrogh D., Moulding D., Nsengimana J., Pittman A., Puig-Butillé J.A., Parmar K., Sebire N.J., Scherer S., Stadnik P., Stanier P., Tell G., Waelchli R., Zarrei M., Puig S., Bataille V., Xing Y., Healy E., Moore G.E., Di W.L., Newton-Bishop J., Downward J, & Kinsler V.A. (2021). Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype. Genetics in Medicine, 23(9), 1636-1647.

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9

Exome Sequencing of Tumor-Normal Pairs

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Exome libraries of matched pairs of tumor / normal genomic DNAs were generated using the Agilent SureSelect XT kit and Agilent Automation Systems NGS system per manufacturer’s instructions. 1 ug of each genomic DNA was sheared using a Covaris E220 to a peak target size of 150 bp. Fragmented DNA was concentrated using AMPureXP beads (Beckman Coulter), and DNA ends were repaired using T4 DNA polymerase, Klenow polymerase, and T4 polynucleotide kinase. 3’ A-tailing with exo-minus Klenow polymerase was followed by ligation of Agilent paired-end oligo adapters to the genomic DNA fragment. Ligated DNA was PCR amplified for 8 cycles and purified using AMPure XP beads and quantitated using the Quant-It BR kit (Invitrogen). 500 ng of sample libraries were hybridized to the Agilent biotinylated SureSelect v4 Exome + UTR Capture Library at 65°C for 72 hr following the manuufacturer’s protocol. The targeted exon fragments were captured on Dynabeads MyOne Strepavidin T1 (Invitrogen), washed, eluted, and enriched by amplification with Agilent post-capture primer and an indexed reverse primer for multiplexing 12 additional cycles. After purification of the PCR products with AMPure XP beads, the quality and quantity of the resulting exome libraries were analyzed using an Agilent Bioanalyzer High Sensitivity chip.

Grasso C.S., Tang Y., Truffaux N., Berlow N.E., Liu L., Debily M.A., Quist M.J., Davis L.E., Huang E.C., Woo P.J., Ponnuswami A., Chen S., Johung T.B., Sun W., Kogiso M., Du Y., Lin Q., Huang Y., Hütt-Cabezas M., Warren K.E., Dret L.L., Meltzer P.S., Mao H., Quezado M., van Vuurden D.G., Abraham J., Fouladi M., Svalina M.N., Wang N., Hawkins C., Nazarian J., Alonso M.M., Raabe E., Hulleman E., Spellman P.T., Li X.N., Keller C., Pal R., Grill J, & Monje M. (2015). Functionally-defined Therapeutic Targets in Diffuse Intrinsic Pontine Glioma: A Report of the Children’s Oncology Group DIPG Preclinical Consortium. Nature medicine, 21(6), 555-559.

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10

Exome Sequencing of Tumor-Normal Pairs

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Exome libraries of matched pairs of tumor / normal genomic DNAs were generated using the Agilent SureSelect XT kit and Agilent Automation Systems NGS system per manufacturer’s instructions. 1 ug of each genomic DNA was sheared using a Covaris E220 to a peak target size of 150 bp. Fragmented DNA was concentrated using AMPureXP beads (Beckman Coulter), and DNA ends were repaired using T4 DNA polymerase, Klenow polymerase, and T4 polynucleotide kinase. 3’ A-tailing with exo-minus Klenow polymerase was followed by ligation of Agilent paired-end oligo adapters to the genomic DNA fragment. Ligated DNA was PCR amplified for 8 cycles and purified using AMPure XP beads and quantitated using the Quant-It BR kit (Invitrogen). 500 ng of sample libraries were hybridized to the Agilent biotinylated SureSelect v4 Exome + UTR Capture Library at 65°C for 72 hr following the manuufacturer’s protocol. The targeted exon fragments were captured on Dynabeads MyOne Strepavidin T1 (Invitrogen), washed, eluted, and enriched by amplification with Agilent post-capture primer and an indexed reverse primer for multiplexing 12 additional cycles. After purification of the PCR products with AMPure XP beads, the quality and quantity of the resulting exome libraries were analyzed using an Agilent Bioanalyzer High Sensitivity chip.

Grasso C.S., Tang Y., Truffaux N., Berlow N.E., Liu L., Debily M.A., Quist M.J., Davis L.E., Huang E.C., Woo P.J., Ponnuswami A., Chen S., Johung T.B., Sun W., Kogiso M., Du Y., Lin Q., Huang Y., Hütt-Cabezas M., Warren K.E., Dret L.L., Meltzer P.S., Mao H., Quezado M., van Vuurden D.G., Abraham J., Fouladi M., Svalina M.N., Wang N., Hawkins C., Nazarian J., Alonso M.M., Raabe E., Hulleman E., Spellman P.T., Li X.N., Keller C., Pal R., Grill J, & Monje M. (2015). Functionally-defined Therapeutic Targets in Diffuse Intrinsic Pontine Glioma: A Report of the Children’s Oncology Group DIPG Preclinical Consortium. Nature medicine, 21(6), 555-559.

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Sureselectxt kit

Lab products found in correlation

18 protocols using sureselectxt kit

HAdV DNA Library Preparation and Sequencing

Whole Exome Sequencing of Mouse Tumors

Targeted Sequencing of M. tuberculosis

NSCLC Tissue Sampling and DNA Extraction

FFPE Tissue DNA Extraction and Exome Sequencing

Whole Exome Sequencing Library Preparation

Targeted Sequencing of DCM Genes

Targeted X-Y Chromosome Sequencing

Exome Sequencing of Tumor-Normal Pairs

Exome Sequencing of Tumor-Normal Pairs

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