Sureselect human all exon v5 utrs 75 mb kit

Manufactured by Agilent Technologies

The SureSelect Human All Exon v5 + UTRs 75 MB kit is a comprehensive genomic solution for target enrichment and sequencing of human protein-coding exons and untranslated regions (UTRs). It is designed to capture the coding regions and UTRs of the human genome in a single reaction.

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Lab products found in correlation

Sureselect post capture indexing forward and index pcr reverse primers, by Agilent Technologies (4 mentions) Hiseq 3000 4000 sequencing kit, by Illumina (3 mentions) Hiseq 3000 4000 pe cluster kit, by Illumina (3 mentions) Rta version 2, by Illumina (3 mentions) Bioanalyzer dna 1000 chip, by Agilent Technologies (2 mentions) Qubit fluorometry, by Thermo Fisher Scientific (2 mentions) Hiseq 4000, by Illumina (2 mentions) Cbot and cbot paired end cluster kit v3, by Illumina (1 mentions) Bravo liquid handler, by Agilent Technologies (1 mentions) Truseq sbs v3 sequencing kit, by Illumina (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Hiseq 2000, by Illumina (1 mentions) Bioanalyzer dna 1000 chip, by Thermo Fisher Scientific (1 mentions) Agilent bravo, by Agilent Technologies (1 mentions) Cbot and hiseq 3000 4000 pe cluster kit, by Illumina (1 mentions)

5 protocols using sureselect human all exon v5 utrs 75 mb kit

Whole Exome Sequencing of Frozen Samples

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Three fresh frozen samples were submitted for whole exome sequencing at Mayo Clinic molecular genomic facility. In brief, paired-end libraries were prepared with 1.0 μg of genomic DNA in accordance with the manufacturer's protocol (Agilent Technologies, Inc, Santa Clara, Calif). Whole-exon capture was performed with 750 ng of the prepped library following the protocol for the SureSelect Human All Exon v5 + UTRs 75 Mb kit (Agilent Technologies, Inc). The purified capture products were then amplified with use of SureSelect Post-Capture Indexing forward and Index polymerase chain reaction reverse primers (Agilent Technologies, Inc) for 12 cycles. Concentration and size distribution of the completed captured libraries were assessed on Qubit (Invitrogen, Waltham, Mass) and Bioanalyzer DNA 1000 chip (Agilent Technologies, Inc). Libraries were sequenced at an average coverage of about 80× in accordance with standard protocol of the cBot and HiSeq 3000/4000 PE Cluster Kit (Illumina, San Diego, Calif). The flow cells were sequenced as 150 × 2 paired end reads on the HiSeq 4000 with the HiSeq 3000/4000 sequencing kit and collection software (HCS version 3.3.52; Illumina). Base calling was performed with Real-Time Analysis version 2.7.3 (Illumina). All procedures were performed in accordance with the manufacturer's instructions.

Liu Y., Bhagwate A., Winham S.J., Stephens M.T., Harker B.W., McDonough S.J., Stallings-Mann M.L., Heinzen E.P., Vierkant R.A., Hoskin T.L., Frost M.H., Carter J.M., Pfrender M.E., Littlepage L., Radisky D.C., Cunningham J.M., Degnim A.C, & Wang C. (2022). Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics. BMC Medical Genomics, 15, 195.

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Whole Exome Sequencing of hiPSC DNA

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Genomic DNA samples from patient-derived cell lines (hiPSCs) were obtained using a DNeasy blood and tissue kit (Qiagen), and whole exome sequencing was carried out at the Mayo Clinic Advanced Genomics Technology Center. Paired-end libraries using 1.0 μg of gDNA were prepared according to the standard manufacturer’s instructions with the Agilent Bravo liquid handler. The concentration and size distribution of the completed libraries was determined using an Agilent DNA 1000 bioanalyser chip and Qubit fluorometry (Invitrogen). Whole exome capture was carried out using 750 ng of the prepped library following the protocol for the SureSelect Human All Exon v5 + UTRs 75 MB kit (Agilent). The purified capture products were then amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. Libraries were sequenced at an average coverage of 80× following Illumina’s standard protocol using the Illumina cBot and cBot paired-end cluster kit v3. The flow cells were sequenced as 101 × 2 paired-end reads on an Illumina HiSeq 2000 using a TruSeq SBS sequencing kit v3 and HCS v2.0.12 data collection software. Base-calling was carried out using Illumina’s RTA v1.17.21.3 (ref. 60 (link)).

Skamagki M., Correia C., Yeung P., Baslan T., Beck S., Zhang C., Ross C.A., Dang L., Liu Z., Giunta S., Chang T.P., Wang J., Ananthanarayanan A., Bohndorf M., Bosbach B., Adjaye J., Funabiki H., Kim J., Lowe S., Collins J.J., Lu C.W., Li H., Zhao R, & Kim K. (2017). ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors. Nature cell biology, 19(9), 1037-1048.

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Whole-Exome Sequencing of Genetic Disorders

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Whole-exome sequencing (WES) was performed on the proband and her parents at a CLIA-certified laboratory (Mayo Clinic) following a standard procedure summarized as follows: Paired-end libraries were prepared following Agilent’s (Santa Clara, CA, USA) protocol. Whole-exon capture was carried out following the protocol for Agilent’s SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products were amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. The concentration and size distribution of the completed captured libraries were determined on Qubit (Invitrogen, Carlsbad, CA, USA) and Agilent Bioa-nalyzer DNA 1000 chip. Sequencing was performed following Illumina’s standard protocol in the Illumina (San Diego, CA, USA) cBot and HiSeq 3000/4000 PE Cluster Kit (average coverage ~80×). The flow cells were sequenced as 150 × 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling was performed using Illumina’s RTA version 2.7.3.

Kotwal A., Ferrer A., Kumar R., Singh R.J., Murthy V., Schultz-Rogers L., Zimmermann M., Lanpher B., Zimmerman K., Stabach P.R., Klee E., Braddock D.T, & Wermers R.A. (2020). Clinical and Biochemical Phenotypes in a Family With ENPP1 Mutations. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 35(4), 662-670.

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Whole Exome Sequencing Workflow

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DNA-sequencing was performed by Mayo Clinic Core facility using Sure Select XT Whole Exon Capture v5 + UTRs 75 MB and Illumina HiSeq 4000 Paired-End Sequencing. Paired-end libraries were prepared using 1.0 μg of genomic DNA following the manufacturer’s protocol (Agilent) using the Agilent Bravo liquid handler. Whole exon capture was carried out using 750 ng of the prepped library following the protocol for Agilent’s SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products are then amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. The concentration and size distribution of the completed libraries was determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced at an average coverage of ~250× following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 150 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kits and HCS v3.3.52 collection software. Base-calling is performed using Illumina’s RTA version 2.7.3.

Zhuang Y., Grainger J.M., Vedell P.T., Yu J., Moyer A.M., Gao H., Fan X.Y., Qin S., Liu D., Kalari K.R., Goetz M.P., Boughey J.C., Weinshilboum R.M, & Wang L. (2021). Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX). NPJ Breast Cancer, 7, 79.

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Trio Whole Exome Sequencing Analysis

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WES from the second miscarriage and both parents was performed at the Clinical Genomics Laboratory (Mayo Clinic). Paired-end libraries were prepared using 1.0 µg of genomic DNA using the Agilent Bravo liquid handler (Agilent) as indicated by the manufacturer. Whole exon capture was carried out using 750 ng of the prepped library following the protocol for Agilent's SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products were amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. Libraries were sequenced at an average coverage of ~80X following Illumina's standard protocol in an Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 150 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling is performed using Illumina's RTA version 2.7.3. Data was processed through an in-house bioinformatics pipeline and analyzed using Ingenuity (Qiagen) by the Center for Individualized Medicine (Mayo Clinic).

Ferrer A., Starosta R.T., Ranatunga W., Ungar D., Kozicz T., Klee E., Rust L.M., Wick M, & Morava E. (2020). Fetal glycosylation defect due to ALG3 and COG5 variants detected via amniocentesis: Complex glycosylation defect with embryonic lethal phenotype. Molecular genetics and metabolism, 131(4).

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