NHGRI/DIR Genomics Core

基因分型使用两种技术中的任何一种进行，Illumina BeadArray用于snp或ABI毛细管电泳测定荧光标记PCR产物的大小，包括STRPs或其他感兴趣的基因组区域。Core采用ABI技术寻找最初由锌指核酸酶（ZFN）技术生成的indels，最近采用CRISPR/Cas诱变方法生成indels。在过去的五年中，Core为多个研究人员和斑马鱼Core的斑马鱼诱变项目处理了120,000个PCR片段。目前，服务包括人类和小鼠SNP基因分型，小鼠，斑马鱼和人类引物测试和（CRISPR/Cas）突变筛选，小鼠速度基因，人类和小鼠STRPs，缺失MLPA，以及用于各种其他应用的片段分析。

Genotyping is performed using either of two technologies, Illumina BeadArray for SNPs or ABI capillary electrophoretic sizing of fluorescently tagged PCR products encompassing STRPs or other genomic regions of interest. The Core adopted ABI technology to find indels generated initially by zinc finger nuclease (ZFN) technology, and recently by CRISPR/Cas mutagenesis approach. The Core processed over the last five years 120,000 PCR fragments for zebrafish mutagenesis projects for multiple investigators and the Zebrafish Core. Currently, services include human and mouse SNP genotyping, mouse, zebrafish and human primer testing and (CRISPR/Cas) mutation screening, mouse speed congenics, human and mouse STRPs, MLPA for deletions, and fragment analysis for a variety of other applications. Both Illumina and ABI technologies are widely used by a large number of NHGRI investigators. This year, a total of 24 investigators including four adjunct faculty representing eight branches and two programs (NISC and UDP) used the Core genotyping services, compared to a total of 21 investigators, last year. Over the past five years (FY2012-FY2016), there has been a steady increase in service requests received (353, 583, 707 853, and 977) and the number of DNA samples processed (9,895, 17,608, 40,350, 42,466 and 48,452). Human SNP genotyping was carried out on eight different BeadArray types using Illumina Infinium technology. We processed, this year, 7,044 human DNA samples and generated 7.74 billion genotypes. Last year (FY2015), these numbers were 6,188 human DNA samples and 2.6 billion genotypes. In addition, this year, 120 samples were processed using human methylation arrays, and 192 using mouse SNP arrays. The genotyping data is used for studies related to Fanconi anemia, Head and Neck cancer, Inherited bone marrow failure syndromes, Smith Magenis syndrome, Non-alcoholic Fatty Liver Disease (NAFLD), Coloboma, iPSCs, Holoprosencephaly, Gaucher's Disease/Parkinson's Disease, Congenital Heart Disease study, ADHD; Twins Study, among others. The data was analyzed for linkage, association, copy number variation, deletion intervals, methylation status, variations introduced by the iPS technology, parent-of-origin of deletions, mosaicism, and uniparental disomy, and to generate haplotypes for discovering variants from sequence data. In addition to numerous small projects, we do have some large SNP projects. Over the last five years (FY2012-FY2016), the number of samples we did SNP genotyping for NISC were 1152, 1536, 2256, 4032 and 2256. We understand that the genotyping samples processed for NISC belong to multiple investigators from other institutes, indicating the Genomics Core serves a larger scientific community than just NHGRI. Further, a large SNP genotyping project started last year (816 DNA samples) was extended to another 2792 DNA samples this year. Samples run on SNP and methylation arrays (7,356 samples) represent only about 15% of the total 48,452 DNA samples processed by the Core this year. The remaining samples (41,096) were processed using ABI technology, which has the capability to separate fluorescently labeled PCR products at single-base resolution. An efficient screening strategy for identification of germline transmitting founder fish and the size of insertion/deletion mutations generated by ZFNs and TALENs, started as a modest effort in 2010, has had a huge surge due to the introduction of CRISPR/Cas technology for targeted mutagenesis. The Genomics Core has processed a large number of zebrafish DNA samples (5,492, 14,619, 35,539, 28,986, and 34,714, respectively for the FY2012-2016), and to a lesser extent, the CRISPR technology is being extended to mouse and human mutagenesis as well. The Core has been assisting investigators with data analysis and access to software/tools, such as GoldenHelix, Nexus, and GenomeStudio. The Core provided training on Illuminas GenomeStudio, of which the Core had purchased unlimited licenses, and several researchers at NHGRI are using this software on their own computers. The yearly renewal expense of Nexus and GoldenHelix limits their availability to only one computer in the Core. The Core helps researchers to take advantage of learning and using these tools, and also helps with the handling, collection, evaluation, and processing of SNP and other data sets. The Core has provided significant data analysis support over the past five years. The services are related to copy number variation, linkage disequilibrium analysis, population stratification, and association studies. Analyses for detecting deletions, duplications, loss of heterozygosity, and regions portraying signs of chromosomal mosaicism in DNA samples from patients diagnosed with Fanconi anemia and head and neck cancer were also performed. Other studies include changes associated with the processing of iPS, Acute myeloid leukemia, Smith-Magenis syndrome, Febrile Infection-Related Epilepsy Syndrome, population stratification and variant frequency ranking of the African-American Ancestral SNPs, and eye diseases Coloboma and Leber Congenital Amaurosis (LCA), ADHD, Congenital Heart Disease, Holoprosencephaly, Turner syndrome, and Fanconi anemia. In addition to performing analysis, the core also helps with troubleshooting or problem solving any issues investigators may have in handling their data. This service is of huge value to investigators with small projects, as are most users of the Core, who do not have the required tools or expertise for the analysis of large data sets. We added three new genotyping panels last year: HumanOmni2.5Exome, African Diaspora, and MEGA Consortium, and this year we added HumanOmni5Exome. These new array types involved a new LCG technology that created a few initial hurdles that needed to be resolved. We did very well in using these chips for genotyping for 3000 DNA samples in the FY2016. The SNP genotyping Infinium assay requires 4 l of 50 ng/l (200ng) DNA, however, we have explored the methodologies that is allowing investigators to get genotyping done at the Core for DNA samples in limited amounts.