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Paired end pre capture libraries

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Paired-end pre-capture libraries are a type of sequencing library preparation method used in genomic research. The core function of these libraries is to generate DNA fragments with known sequence on both ends, which allows for more accurate mapping and analysis of the sequenced regions.

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Lab products found in correlation

Multiplexing sampleprep guide 1005361 d, by Illumina (3 mentions) Kapa hifi dna polymerase, by Roche (2 mentions) Ampure xp bead, by Beckman Coulter (1 mentions) E220 system, by Covaris (1 mentions) Hiseq 2000 genome analyzer, by Illumina (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions)

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Paired-end libraries Pre-capture libraries

6 protocols using paired end pre capture libraries

1

Illumina Barcoded Paired-End Capture Library Preparation

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Genomic DNA samples were constructed into Illumina paired-end pre-capture libraries according to the manufacturer’s protocol (Illumina Multiplexing_SamplePrep_Guide_1005361_D) with modifications as described in the BCM-HGSC Illumina Barcoded Paired-End Capture Library Preparation protocol that is accessible from the HGSC website (https://hgsc.bcm.edu/sites/default/files/documents/Illumina_Barcoded_Paired-End_Capture_Library_Preparation.pdf).
Pickering C.R., Zhou J.H., Lee J.J., Drummond J.A., Peng S.A., Saade R.E., Tsai K.Y., Curry J.L., Tetzlaff M.T., Lai S.Y., Yu J., Muzny D.M., Doddapaneni H., Shinbrot E., Covington K.R., Zhang J., Seth S., Caulin C., Clayman G.L., El-Naggar A.K., Gibbs R.A., Weber R.S., Myers J.N., Wheeler D.A, & Frederick M.J. (2014). Mutational landscape of aggressive cutaneous squamous cell carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(24), 6582-6592.
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2

Illumina Paired-End Library Preparation

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Libraries were constructed into Illumina paired-end pre-capture libraries according to the manufacturer's protocol (Illumina Multiplexing_SamplePrep_Guide_1005361_D) with modifications as described in the BCM-HGSC protocol (https://www.hgsc.bcm.edu/content/protocols-sequencing-library-construction).
Miszalski-Jamka K., Jefferies J.L., Mazur W., Głowacki J., Hu J., Lazar M., Gibbs R.A., Liczko J., Kłyś J., Venner E., Muzny D.M., Rycaj J., Białkowski J., Kluczewska E., Kalarus Z., Jhangiani S., Al-Khalidi H., Kukulski T., Lupski J.R., Craigen W.J, & Bainbridge M.N. (2017). Novel Genetic Triggers and Genotype-Phenotype Correlations in Patients with Left Ventricular Non-Compaction. Circulation. Cardiovascular genetics, 10(4), e001763.
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3

FFPE DNA Library Preparation Protocol

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Tumor DNA samples were predominantly derived from FFPE except 3 ovarian samples, which were fresh frozen (FF); and Normal DNA samples, which were derived from the peripheral blood. DNA samples were constructed using Illumina paired-end pre-capture libraries following the protocol previously described for FF samples(Rokita et al., 2019 (link)). DNA input for libraries was 250 ng of DNA and Pre-capture Ligation Mediated-PCR (LM-PCR) was performed for 8-10 cycles using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc., Cat # KK2612)
Wheeler D.A., Takebe N., Hinoue T., Hoadley K.A., Cardenas M.F., Hamilton A.M., Laird P.W., Wang L., Johnson A., Dewal N., Miller V., Piñeyro D., de Moura M.C., Esteller M., Shen H., Zenklusen J.C., Tarnuzzer R., McShane L.M., Tricoli J.V., Williams P.M., Lubensky I., O’Sullivan-Coyne G., Kohn E.C., Little R.F., White J., Malik S., Harris L., Weil C., Chen A.P., Karlovich C., Rodgers B., Shankar L., Jacobs P., Nolan T., Hu J., Muzny D.M., Doddapaneni H., Korchina V., Gastier-Foster J., Bowen J., Leraas K., Edmondson E.F., Doroshow J.H., Conley B.A., Ivy S.P, & Staudt L.M. (2020). Molecular Features of Cancers Exhibiting Exceptional Responses to Treatment. Cancer cell, 39(1), 38-53.e7.
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4

FFPE DNA Illumina Library Preparation

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FFPE DNA samples were constructed into Illumina paired-end pre-capture libraries according to the 500 ng or 250 ng (depending on sample quantity availability) of DNA in 50ul volume. gDNA was sheared into fragments of the average size of 250–300 base pairs in a Covaris plate with an E220 system (Covaris, Inc. Woburn, MA) followed by end-repair (NEBNext End Repair Module—E6050L) and A-tailing (NEBNext dA-Tailing Module—E6053L), ligated with Illumina Dual index PE adapter using (Life Tech. ExpressLink Ligase- A13726101), Pre-capture Ligation Mediated-PCR (LM-PCR) was performed for 6–8 cycles using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc., Cat # KK2612). Universal P1.1 (5′-AATGATACGGCGACCACCGAGA) and P3 (5′-CAAGCAGAAGACGGCATACGAGA) primers were used to amplify the ligated products. AMPure XP beads (A63882, Beckman Coulter) were used for library purification. Quantification and size distribution of the pre-capture LM-PCR product were determined using the Fragment Analyzer capillary electrophoresis system (Agilent Technologies, Inc).
Lim H., Gingras M.C., Zhao J., Byun J., Castro P.D., Tsavachidis S., Hu J., Doddapaneni H., Han Y., Muzny D.M., Gibbs R.A., Amos C.I, & Thrift A.P. (2024). Somatic mutations of esophageal adenocarcinoma: a comparison between Black and White patients. Scientific Reports, 14, 8988.
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5

Illumina Pre-Capture Library Preparation

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DNA samples were constructed into Illumina paired-end pre-capture libraries according to the manufacturer’s protocol (Illumina Multiplexing_SamplePrep_Guide_1005361_D). The complete library and capture protocol, as well as oligonucleotide sequences have been described in detail previously [15 (link)]. For exome capture, each library pool was hybridized in solution to the BCM-HGSC designed VCRome 2.1 capture reagent according to the manufacturer’s protocol (NimbleGen) with minor revisions.
Liu Y., Kheradmand F., Davis C.F., Scheurer M.E., Wheeler D., Tsavachidis S., Armstrong G., Simpson C., Mandal D., Kupert E., Anderson M., You M., Xiong D., Pikielny C., Schwartz A.G., Bailey-Wilson J., Gaba C., De Andrade M., Yang P., Pinney S.M., Amos C.I, & Spitz M.R. (2016). Focused Analysis of Exome Sequencing Data for Rare Germline Mutations in Familial and Sporadic Lung Cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 11(1), 52-61.
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6

Whole Exome Sequencing of Tumor and Normal Samples

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DNA was isolated from tumor specimens, and normal DNA was isolated from leukocytes in peripheral blood. Whole exome sequencing was performed on DNA from all 27 blood and tumor samples. We extracted the DNA from the paired tumor and peripheral blood leukocytes using QIAamp DNA Mini Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA). Genomic DNA samples were constructed into Illumina paired-end precapture libraries and prepared using protocols recommended by Illumina. Captured DNA libraries were sequenced with the Illumina HiSeq 2000 Genome Analyzer to an average coverage of 144X, yielding 150 (2 × 75) base pairs from the final library fragments. Reads were aligned to the hg19 (GRCh37) build using the Burrows-Wheeler Aligner (BWA)(16 (link)), and then Genome Analysis Toolkit (GATK) (17 (link)) was used for base quality score recalibration, indel realignment, and duplicate read removal. The MuTect algorithm (18 ) was used to identify somatic single nucleotide variants (SNVs) in whole-exome sequencing data. MuTect identifies candidate somatic SNVs by Bayesian statistical analysis of bases and their qualities in the tumor and normal BAM files at a given genomic locus.
Cho S.J., Yoon C., Lee J.H., Chang K.K., Lin J.X., Kim Y.H., Kook M.C., Aksoy B.A., Park D.J., Ashktorab H., Smoot D.T., Schultz N, & Yoon S.S. (2018). KMT2C mutations in diffuse type gastric adenocarcinoma promote epithelial-to-mesenchymal transition. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(24), 6556-6569.
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