The integration site of the BAC in the bovine genome by chromosomal walking were unsuccessful (data not shown), suggesting a break in the BAC; thus, the endogenous sequences flanking the integration site and whether there was integration of multiple copies of the transgene remained unknown. Therefore, an efficient method for identifying the specific transgene integration sites was needed. Next-generation sequencing has had a profound impact on genomic research and has become a powerful tool with a diverse range of applications. Next-generation sequencing has enabled the comprehensive analysis of whole genomes in a costeffective, routine and widespread manner [17]. In this study, we investigated the use of next-generation sequencing and subsequent bioinformatic Title Loaded From File analyses to characterize the sequence signature of the hLF BAC transgene and determine the exact insertion site(s) and copy number in three individual transgenic cattle.DNA Mini kit (Qiagen, German) and stored at ?0uC until needed. This animal work was approved by the Institutional Animal Care and Use Committee of China Agricultural University (ID: SKLAB-2010-05-01). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Whole Genome SequencingDNA was extracted from the blood of the three transgenic cattle with a QIAGEN DNA extraction kit (Qiagen Sciences, Germantown, MD). A total of 1.5 mg whole genomic DNA was sonicated with a Bioruptor sonication system (Diagenode, Inc.) to produce fragments ranging in size from 250 to 650 bp with a peak at 300?400 bp. DNA in 0.5 6 TE buffer was pulsed for 21 cycles; each cycle was performed at 30 sec on and 30 sec off under high frequency. The DNA fragments were purified with a Qiagen purification kit (Qiagen Sciences, Germantown, MD). The DNA fragments were blunt end repaired and adenylated, followed by adaptor ligation according to the protocol of the Truseq DNA sample preparation kit V2 (Illumina, San Diego, CA). Size selection was performed on a 2 agarose gel. The portion of the gel corresponding to 450?50 bp DNA was excised and purified with a Qiagen gel extraction kit (Qiagen Sciences, Germantown, MD). PCR enrichment was performed for 10 cycles, followed by purification. The libraries were quantified by a LightCyclerH 480|Real-Time PCR System (Roche Diagnostics), and the insert size was measured with an Agilent 2100 (Agilent Technologies, San Diego CA). Massive parallel sequencing of the DNA libraries was applied to cBot and Hiseq2000 according to the manufacturer’s protocols (Illumina, San Diego CA). The read numbers collected for cattle #040825, #050211 and #101026 were 264 M, 246 M and 307 M, respectively.Materials and Methods Transgenic AnimalsThe generation of transgenic cattle that specifically express human 1527786 lactoferrin (hLF) in milk has been described previously [14]. hLF BAC clones containing the entire hLF genomic sequence (GenBank accession number: U95626) were obtained by screening a human BAC Title Loaded From File library (Genome Systems Inc.). Three transgenic cattle were detected, including the transgenic founders (F0) #040825 and #050211, which were cloned from the same fetal fibroblast cell lines, and #101026, from the second generation (F2) of #040825. Genomic DNA was extracted from ear biopsies of the three transgenic cattle with a QIAsymphonyData AnalysisSequencing depth analysis was performed to estimate the copy numbers of the BAC and pBeloBAC vector. Briefly, lowquality reads wer.The integration site of the BAC in the bovine genome by chromosomal walking were unsuccessful (data not shown), suggesting a break in the BAC; thus, the endogenous sequences flanking the integration site and whether there was integration of multiple copies of the transgene remained unknown. Therefore, an efficient method for identifying the specific transgene integration sites was needed. Next-generation sequencing has had a profound impact on genomic research and has become a powerful tool with a diverse range of applications. Next-generation sequencing has enabled the comprehensive analysis of whole genomes in a costeffective, routine and widespread manner [17]. In this study, we investigated the use of next-generation sequencing and subsequent bioinformatic analyses to characterize the sequence signature of the hLF BAC transgene and determine the exact insertion site(s) and copy number in three individual transgenic cattle.DNA Mini kit (Qiagen, German) and stored at ?0uC until needed. This animal work was approved by the Institutional Animal Care and Use Committee of China Agricultural University (ID: SKLAB-2010-05-01). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Whole Genome SequencingDNA was extracted from the blood of the three transgenic cattle with a QIAGEN DNA extraction kit (Qiagen Sciences, Germantown, MD). A total of 1.5 mg whole genomic DNA was sonicated with a Bioruptor sonication system (Diagenode, Inc.) to produce fragments ranging in size from 250 to 650 bp with a peak at 300?400 bp. DNA in 0.5 6 TE buffer was pulsed for 21 cycles; each cycle was performed at 30 sec on and 30 sec off under high frequency. The DNA fragments were purified with a Qiagen purification kit (Qiagen Sciences, Germantown, MD). The DNA fragments were blunt end repaired and adenylated, followed by adaptor ligation according to the protocol of the Truseq DNA sample preparation kit V2 (Illumina, San Diego, CA). Size selection was performed on a 2 agarose gel. The portion of the gel corresponding to 450?50 bp DNA was excised and purified with a Qiagen gel extraction kit (Qiagen Sciences, Germantown, MD). PCR enrichment was performed for 10 cycles, followed by purification. The libraries were quantified by a LightCyclerH 480|Real-Time PCR System (Roche Diagnostics), and the insert size was measured with an Agilent 2100 (Agilent Technologies, San Diego CA). Massive parallel sequencing of the DNA libraries was applied to cBot and Hiseq2000 according to the manufacturer’s protocols (Illumina, San Diego CA). The read numbers collected for cattle #040825, #050211 and #101026 were 264 M, 246 M and 307 M, respectively.Materials and Methods Transgenic AnimalsThe generation of transgenic cattle that specifically express human 1527786 lactoferrin (hLF) in milk has been described previously [14]. hLF BAC clones containing the entire hLF genomic sequence (GenBank accession number: U95626) were obtained by screening a human BAC library (Genome Systems Inc.). Three transgenic cattle were detected, including the transgenic founders (F0) #040825 and #050211, which were cloned from the same fetal fibroblast cell lines, and #101026, from the second generation (F2) of #040825. Genomic DNA was extracted from ear biopsies of the three transgenic cattle with a QIAsymphonyData AnalysisSequencing depth analysis was performed to estimate the copy numbers of the BAC and pBeloBAC vector. Briefly, lowquality reads wer.
