The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery.

Single nucleotide polymorphisms (SNPs) in the KLK3 gene on chromosome 19q13.33 are associated with serum prostate-speciWc antigen (PSA) levels. Recent genome wide association studies of prostate cancer have yielded conXicting results for association of the same SNPs with prostate cancer risk. Since the KLK3 gene encodes the PSA protein that forms the basis for a widely used screening test for prostate cancer, it is critical to fully characterize genetic variation in this region and assess its relationship with the risk of prostate cancer. We have conducted a nextgeneration sequence analysis in 78 individuals of European ancestry to characterize common (minor allele frequency, MAF >1%) genetic variation in a 56 kb region on chromosome 19q13.33 centered on the KLK3 gene (chr19:56,019,829-56,076,043 bps). We identiWed 555 polymorphic loci in the process including 116 novel SNPs and 182 novel insertion/deletion polymorphisms (indels). Based on tagging analysis, 144 loci are necessary to tag the region at an r 2 threshold of 0.8 and MAF of 1% or higher, while 86 loci are required to tag the region at an r 2 threshold of 0.8 and MAF >5%. Our sequence data augments coverage by 35 and 78% as compared to variants in dbSNP and HapMap, respectively. We observed six non-synonymous amino acid or frame shift changes in the KLK3 gene Electronic supplementary material The online version of this article

Characterization of the somatic sequence variations that accrue in cells is critical for understanding the pronounced cellular and clinical heterogeneity observed in cancer. The ability to efficiently detect variations in tumors can help to identify biomarkers which may be relevant to clinical trials, support more accurate prognosis, and help guide more effective choices of therapy. Next-generation sequencing (NGS) has become a valuable tool for discovering somatic mutations in cancers. Here we present an alternative approach to current methodologies for addressing these needs. HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) is a targeted NGS method based on optimized, multiplexed molecular inversion probes (MIPs). This is a convenient, sensitive and cost-effective target enrichment technology for SNP discovery and SNP validation in cancer-related genes. HEAT-Seq probes target both DNA strands and were designed to facilitate bioinformatic error correction. Molecule identifiers (UIDs) incorporated into the probes tag PCR duplicates and support ultra-sensitive detection of low frequency variants, reduction of false positives, and accurate assessment of molecular complexity free of amplification bias. Sensitivities for allele detection have been measured to below 1%. The HEAT-Seq workflow from input DNA to sequence-ready sample can be completed in ≤8 hours without any requirement for a separate library preparation step, which saves both time and cost. Additionally, the capture, amplification and sample clean up steps are performed in a single reaction tube. This eliminates the need to transfer samples to subsequent reaction tubes, preserves sample identity, prevents cross contamination, limits sample loss, and makes the HEAT-Seq protocol easy to automate. A comparison of current PCR-based targeted sequencing with the new HEAT-Seq technology indicates significant advantages for HEAT-Seq in the accurate quantification of low frequency cancer variants. In summary, HEAT-Seq technology offers a rapid, convenient, automatable option to identify genomic DNA variants and enable advances in cancer research. Citation Format: Keynttisha Jefferson, Heather Halvensleben, Dawn Green, Ryan Bannen, Michael Brockman, Todd Richmond, Daniel Burgess. A novel molecular inversion probe (MIP) system for the streamlined identification of germline and somatic sequence variants in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5223.

Next generation sequencing (NGS) of enriched targets is increasingly being used to discover and track mutations in genes implicated in cancer. Amplicon-based target enrichment approaches are frequently used with cancer samples because there is no sample library preparation needed and primer-based approaches are typically efficient with sequencing reads. However, traditional amplicon approaches don't scale up well and may have PCR and coverage uniformity bias. As the number of samples is increased, a fast, reliable, and cost-effective target enrichment method is a critical tool for mutation discovery and tracking. Existing target enrichment approaches based on molecular inversion probes (MIPs) have presented challenges with probe design and dependability. We have developed HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) using advanced versions of MIPs which enable library-free enrichment, improved scalability, and the ability to remove PCR duplicates. We have also developed companion command line software which trims primer sequences and removes PCR duplicates. This software uses community standard file formats (FASTQ, SAM/BAM) and is intended to be compatible with common “best practice” NGS analysis pipelines. Here we describe two HEAT-Seq cancer panels which have been optimized to improve coverage uniformity and probe reliability. The HEAT-Seq Oncology Panel targets both strands for all protein coding bases in 60 cancer-related genes where possible. The target for this design is 245 kb. With 2 million 2×76bp reads for each of 8 replicates, we observe an on-target read rate of >95%. After duplicate removal, the% of target bases with at least 20x coverage is >90%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼90%. The HEAT-Seq Ultra Hot-Spot Panel is a mutation-focused design that provides ultra-deep sequencing coverage capability for sequencing of mutation hot-spots in 53 cancer-related genes to detect low frequency variants in heterogeneous samples. The target for this design is 30.5 kb. With 500,000 2×76bp reads for each of 24 replicates, we observe an on-target read rate of ∼80%. After duplicate removal, the% of target bases with at least 20x coverage is >95%. We observe uniformity (percent of probes > = 20% of the target mean) of ∼95%. We also observed detection of validated low frequency variants down to 1%. In summary, both panels have been shown to capture cancer-related genes and target regions at depths sufficient to identify and track genomic variants. Citation Format: Ryan Bannen, Michael Brockman, Mark D’Ascenzo, Keynttisha Jefferson, Dawn Green, Heather Halvensleben, Kurt Heilman, Todd Richmond, Daniel Burgess. Cancer target enrichment panels using advanced molecular inversion probes (MIPs) with ability to reduce amplification bias and detect low frequency variants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5217.

Faithful annotation of tissue-specific transcript isoforms is important not only to understand how genes are organized and regulated but also to identify potential novel, unannotated exons of genes, which may be additional targets of mutation in disease states or while performing mutagenic screens. We have developed a microarray enrichment methodology followed by long-read, next-generation sequencing for identification of unannotated transcript isoforms expressed in two Drosophila tissues, the ovary and the testis. Even with limited sequencing, these studies have identified a large number of novel transcription units, including 5′ exons and extensions, 3′ exons and extensions, internal exons and exon extensions, gene fusions, and both germline-specific splicing events and promoters. Additionally, comparing our capture dataset with tiling array and traditional RNA-seq analysis, we demonstrate that our enrichment strategy is able to capture low-abundance transcripts that cannot readily be identified by the other strategies. Finally, we show that our methodology can help identify transcriptional signatures of minority cell types within the ovary that would otherwise be difficult to reveal without the CoNECT enrichment strategy. These studies introduce an efficient methodology for cataloging tissue-specific transcriptomes in which specific classes of genes or transcripts can be targeted for capture and sequence, thus reducing the significant sequencing depth normally required for accurate annotation.