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Sureselectxt mouse all exon kit

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The SureSelectXT Mouse All Exon kit is a laboratory equipment product designed for targeted sequencing of the entire mouse exome. It provides a comprehensive solution for capturing and enriching mouse exonic regions to enable efficient and accurate analysis of genetic variations.

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Close spelling variants of this product

SureSelectXT kit (18 citations) SureSelectXT Mouse All Exon (7 citations) SureSelectXT mouse exon kit (2 citations)

42 protocols using sureselectxt mouse all exon kit

1

Whole Exome Sequencing to Identify Mutations

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The whole exome sequencing (WES) was carried out to identify candidate mutations in the exomes of genes. Genomic DNA was isolated from mice ear and BM cells including wildtype and the diseased Hottip-Tg mice, and genomic exome library was captured and constructed according to SureSelectXT Mouse All Exon kit (Agilent, Part Number:5190–4641), and then 100 bp paired-end sequencing was performed using an Illumina NovaSeq S1. Raw sequencing reads were processed through cutadapt (http://cutadapt.readthedocs.io, version 1.2.0) to remove adaptors and low quality reads. These clean reads were mapped to the whole mouse genome (mm10) using BWA with the default settings (bwa/0.7.4) (Liang et al., 2009 (link)). The PCR duplicates were removed with Picard with default parameters (version 1.88) (http://broadinstitute.github.io/picard/), recalibrated with GATK with default setting (version 3.7) (McKenna et al., 2010 (link)), and compared the variance between wildtype and Hottip-Tg group with Strelka (version 2.9.2, default setting)(Kim et al., 2018 (link)), and then variant bases were annotated with SnpEff (latest version) (http://snpeff.sourceforge.net/SnpEff_manual.html) (Cingolani et al., 2012 (link)). All genomics datasets were deposited in the NCBI GEO under accession number (GSE114981).
Luo H., Zhu G., Xu J., Lai Q., Yan B., Guo Y., Fung T.K., Zeisig B.B., Cui Y., Zha J., Cogle C., Wang F., Xu B., Yang F.C., Li W., So C.W., Qiu Y., Xu M, & Huang S. (2019). HOTTIP lncRNA Promotes Hematopoietic Stem Cell Self-Renewal Leading to AML-like Disease in Mice. Cancer cell, 36(6), 645-659.e8.
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2

Whole-Exome Sequencing of Mouse Tumors

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Whole-exome sequencing was performed on extracted DNA from the tumor-derived cell lines. Enrichment for sequencing was performed using the SureSelectXT Mouse All Exon kit (Agilent) following the manufacturer's protocol. Exon-enriched libraries were subjected to paired-end sequencing, 2×100 base pairs read length and 60× minimum coverage.
Raw sequencing reads were aligned to the respective mouse reference genome (NCBI37/mm9). Alignment was performed with the BWA aligner. Concordant read-pairs were identified as potential PCR duplicates and subsequently masked in the alignment file. Somatic mutations and copy number alterations were determined using our in-house analysis pipeline (Peifer et al., 2012 (link); Fernandez-Cuesta et al., 2014 (link)). Data are available on the University of Cologne Scientific Dataset public repository website at https://uni-koeln.sciebo.de/s/JJdOLcanGNN7U92.
Valencia K., Sainz C., Bértolo C., de Biurrun G., Agorreta J., Azpilikueta A., Larrayoz M., Bosco G., Zandueta C., Redrado M., Redín E., Exposito F., Serrano D., Echepare M., Ajona D., Melero I., Pio R., Thomas R., Calvo A, & Montuenga L.M. (2022). Two cell line models to study multiorganic metastasis and immunotherapy in lung squamous cell carcinoma. Disease Models & Mechanisms, 15(1), dmm049137.
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3

Whole Genome Sequencing of Mouse Exome

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Genomic DNA was fragmented using an S2 sonicator (Covaris, USA). Whole genome libraries were constructed using a NEBNext Ultra DNA Library kit (New England Biosciences, Ipswich, MA) with local modifications (specifically Ion Torrent sequencing adaptors were added) and the exome was enriched using a SureSelectXT Mouse All Exon kit (Agilent, Mulgrave, Vic, Australia) that targets 49.6 Mb of the murine genome (221,784 exons across 24,306 coding genes). Sequencing was performed using a Proton sequencer (Ion Torrent, Thermofisher Scientific) system according to manufacturer’s instructions at the Lotterywest State Biomedical Facility Genomics (Nedlands, WA, Australia). Two samples were pooled and sequenced on a P1 chip for 520 flows to an average depth of 42×, which resulted in 80% of the targeted regions being covered with at least an average depth of 13.2× (See Additional file 1).
Sneddon S., Patch A.M., Dick I.M., Kazakoff S., Pearson J.V., Waddell N., Allcock R.J., Holt R.A., Robinson B.W, & Creaney J. (2017). Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma. BMC Cancer, 17, 396.
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4

Targeted Exome Sequencing of Mutant Mice

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DNA extraction from the spleens was performed using ProteinaseK, RNaseA, CellLysis Solution, Protein Precipitation Solution, and DNA Hydration Solution from Qiagen according to the manufacturer’s manual (Qiagen, Venlo,the Netherlands). In-solution targeted enrichment of exonic sequences from both the phenotypically mutant and the control wild-type littermate mouse using the SureSelectXT Mouse All Exon kit (Agilent, Santa Clara, CA, USA) was performed. Libraries were sequenced as 100 bp paired-end runs on a HiSeq 2000 system (Illumina, San Diego, CA, USA). Read alignment to mouse genome assembly mm9 was done with Burrows-Wheeler Aligner (BWA, version 0.5.9), and a total of 10 and 9.2 Gb of mapped sequence data corresponding to an average coverage of 120× (>95 % of the target being covered >20×) and 113× for the mutant and control, respectively, were yielded. Single-nucleotide variants (SNVs) and small insertions and deletions (indels) were detected with SAMtools (v. 0.1.7).
Sabrautzki S., Sandholzer M.A., Lorenz-Depiereux B., Brommage R., Przemeck G., Vargas Panesso I.L., Vernaleken A., Garrett L., Baron K., Yildirim A.O., Rozman J., Rathkolb B., Gau C., Hans W., Hoelter S.M., Marschall S., Stoeger C., Becker L., Fuchs H., Gailus-Durner V., Klingenspor M., Klopstock T., Lengger C., Stefanie L., Wolf E., Strom T.M., Wurst W, & de Angelis M.H. (2016). Viable EdnraY129F mice feature human mandibulofacial dysostosis with alopecia (MFDA) syndrome due to the homologue mutation. Mammalian Genome, 27(11), 587-598.
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5

Mouse Exome Library Preparation and Sequencing

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Mouse exome library preparation was performed using the Agilent SureSelectXT Mouse All Exon kit with modifications. Furthermore, 2 × 100 bp sequencing with a 6‐bp index read was performed using the TruSeq SBS Kit v3 on the HiSeq 2500 (Illumina). Fastq files were generated using BcltoFastq 1.8.4 (Illumina). BWA version 0.7.4 was used to align sequence data to the mouse reference genome (GRCm38.71). Human translocation t(11;14)(q13;q11) bone marrow samples were obtained after informed consent within the AIEOP‐ BFM‐ ALL study and processed via the Macrogen Europe platform, Heidelberg.
García‐Ramírez I., Bhatia S., Rodríguez‐Hernández G., González‐Herrero I., Walter C., González de Tena‐Dávila S., Parvin S., Haas O., Woessmann W., Stanulla M., Schrappe M., Dugas M., Natkunam Y., Orfao A., Domínguez V., Pintado B., Blanco O., Alonso‐López D., De Las Rivas J., Martín‐Lorenzo A., Jiménez R., García Criado F.J., García Cenador M.B., Lossos I.S., Vicente‐Dueñas C., Borkhardt A., Hauer J, & Sánchez‐García I. (2018). Lmo2 expression defines tumor cell identity during T‐cell leukemogenesis. The EMBO Journal, 37(14), e98783.
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6

Preparation of Murine Exome and Whole-Genome Libraries

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Exome libraries and whole-genome libraries were prepared using a modified protocol49 (link). Modifications included: the use of 1,000 ng of treated gDNA, performing only six cycles of PCR amplification and usage of the Agilent SureSelectXT Mouse All Exon Kit for exon target capture. For murine WGS, after adapter ligation, libraries were only amplified by two cycles of PCR. Equimolar quantities of the whole-genome indexed libraries were multiplexed, with 18 libraries per pool. Results from 13 of the 18 libraries were used in our analysis. All pooled libraries were sequenced on an Illumina NovaSeq6000 using the 150-base pair (bp) paired-end format.
Perelli L., Carbone F., Zhang L., Huang J.K., Le C., Khan H., Citron F., Del Poggetto E., Gutschner T., Tomihara H., Soeung M., Minelli R., Srinivasan S., Peoples M., Lam T.N., Lundgren S., Xia R., Zhu C., Mohamed A.M., Zhang J., Sircar K., Sgambato A., Gao J., Jonasch E., Draetta G.F., Futreal A., Bakouny Z., Van Allen E.M., Choueiri T., Signoretti S., Msaouel P., Litchfield K., Turajlic S., Wang L., Chen Y.B., Di Natale R.G., Hakimi A.A., Giuliani V., Heffernan T.P., Viale A., Bristow C.A., Tannir N.M., Carugo A, & Genovese G. (2023). Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer. Nature Cancer, 4(7), 984-1000.
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7

Exome Sequencing of Mouse Samples

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SureSelectXT Mouse All Exon Kit (G7550E-001, Agilent, CA, USA) was used to capture exome sequences according to the standard protocols. The products of exome capture should meet the criteria of 300 ± 30 bp length of fragments and total amount >600 ng. After then, the index-tagged samples were pooled and sequenced on Illumina HiSeq 2000. Burrows-Wheeler Alignment (v0.7.12) was applied to align the reads to the reference genome (mm10) with default parameters, which were then sorted and the duplicated reads were marked by picard-tools (v1.8). Indel realignment and base quality score recalibration were performed with GenomeAnalysisTK (v3.5) using mm10 dbsnp database as known sites. Single-nucleotide polymorphisms and indels were identified by GenomeAnalysisTK HaplotypeCaller (v3.5) with default parameters. Whole-exome sequencing raw data were submitted to SRA database (SRA; http://trace.ncbi.nlm.nih.gov/Traces/sra/, accession number SRP162613)
Yen Y.T., Chien M., Wu P.Y., Ho C.C., Ho C.T., Huang K.C., Chiang S.F., Chao K.S., Chen W.T, & Hung S.C. (2021). Protein phosphatase 2A inactivation induces microsatellite instability, neoantigen production and immune response. Nature Communications, 12, 7297.
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8

Whole Exome Sequencing of Mouse Tumors

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WES was performed on macroscopic GPP53 and GPN1 tumors, and on matched liver samples. All sequencing and analyses were performed by Novogene. Briefly, 1 μg of DNA was used for library preparation using the Agilent SureSelectXT Mouse All Exon kit. Fragmentation was performed to generate 180–280 bp fragments and assessed on the Agilent Bioanalyzer 2100 system for quality control. Captured libraries were sequenced using Illumina NovaSeq 6000. Reads were aligned with Burrows-Wheeler Aligner (BWA; v0.7.17) using the mm10 reference genome. Conversion to BAM files was performed using Picard (v2.18.9). Single-nucleotide variants (SNVs) and InDels were identified by GATK (v4.0), followed by ANNOVAR to annotate variants. Somatic SNVs and InDels were identified by MuTect and Strelka, respectively. Somatic copy number variants (CNVs) were called by Control-FREEC (v11.4), using the setting minCNAlength parameter = 2. Low confidence CNV changes annotated as “genomic superduplications” with CNV = 1 or 3 were omitted from analyses. CNV plots were generated using CNVkit with default settings.
Trieu K.G., Tsai S.Y., Eberl M., Ju V., Ford N.C., Doane O.J., Peterson J.K., Veniaminova N.A., Grachtchouk M., Harms P.W., Swartling F.J., Dlugosz A.A, & Wong S.Y. (2022). Basal cell carcinomas acquire secondary mutations to overcome dormancy and progress from microscopic to macroscopic disease. Cell reports, 39(5), 110779.
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9

Exome Sequencing of Mouse Tumor and Healthy Cells

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DNA from the tumor cell line and from liver cells of a healthy eight-week-old female BALB/c mouse was extracted using the QIAmp DNA Mini Kit (Qiagen). Exome capture was performed with the use of the SureSelectXT Mouse All-Exon kit (Agilent Technologies). Exome libraries were enriched using the SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library protocol for 3 μg DNA (Version B4, August 2015, Agilent Technologies) and sequenced on the HiSeq 2500 v4 platform (Illumina). Sequence reads were aligned to the mm10/GRCm38 genome using Bowtie 2 version 2.2.6 (18 ) (options: no-mixed, no-discordant, very-sensitive). The average on-target coverage was 159x with approximately 93% of the target sequence being covered by at least 30 reads. GATK v3.5 (19 (link)) was used for base quality recalibration (known sites from dbSNP v137), variant calling/genotyping (HaploTypeCaller, GATK) and variant filtering. We used a hard-filtering strategy with the following criteria: DP>20, FS<30, QD>2. The resulting variants were annotated with snpEff version 4.1l (20 (link)) and only variants with impact levels ‘moderate’ and ‘high’ were kept. Finally, variants that were also detected in wildtype BALB/c were removed from the candidate list.
Probst P., Kopp J., Oxenius A., Colombo M.P., Ritz D., Fugmann T, & Neri D. (2017). Sarcoma eradication by doxorubicin and targeted TNF relies upon CD8+ T cell recognition of a retroviral antigen. Cancer research, 77(13), 3644-3654.
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10

Exome Sequencing from DNA Samples

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DNA was purified from samples using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The exome library was prepared using the Agilent SureSelectXT Mouse All Exon Kit with some modifications. Exome capture was performed by hybridization to an RNA library according to the manufacturer’s protocol. Then, the captured library was purified and enriched by binding to MyOne Streptavidin T1 Dynabeads (Life Technologies) and posterior off-bead PCR amplification in the linear range. Sequencing (2 × 100 bp) was carried out in a HiSeq2500 (Illumina) using the TruSeq SBS Kit v3 with a 6-bp index read.
Rodríguez-Hernández G., Opitz F.V., Delgado P., Walter C., Álvarez-Prado Á.F., González-Herrero I., Auer F., Fischer U., Janssen S., Bartenhagen C., Raboso-Gallego J., Casado-García A., Orfao A., Blanco O., Alonso-López D., Rivas J.D., Tena-Dávila S.G., Müschen M., Dugas M., Criado F.J., Cenador M.B., Vicente-Dueñas C., Hauer J., Ramiro A.R., Sanchez-Garcia I, & Borkhardt A. (2019). Infectious stimuli promote malignant B-cell acute lymphoblastic leukemia in the absence of AID. Nature Communications, 10, 5563.
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