Sequence Capture Approach Research Articles

Abstract LB-68: Identification of circulating HPV DNA in urine with a dual sequence capture approach.

Abstract HPV testing is increasingly used for cervical cancer screening in conjunction with cervical cytology. Privacy, cultural, and infrastructure issues challenge the effective implementation of HPV testing in cervical cancer screening. The detection of circulating HPV DNA from urine can be useful for cervical cancer screening. We captured and sequenced the HPV genome after isolating HPV Transrenal DNA (HPV-TrDNA) and then developed a qPCR-based HPV-TrDNA assay to quantify high risk HPV TrDNA. HPV TrDNA molecules are relatively short, fragmented, pieces of circulating DNA (less than 250 nucleotides) that get filtered by the kidneys and can be isolated from urine. Methods: We adsorbed cell-free nucelic acids from urine samples (10mL) of 41 women with 23 LGL and 18 HGL cervical lesions and 31 women with normal cytology on a Q-Sepharose resin followed by a silica-based method. A custom-designed pool of HPV-specific dual sequence capture probes was used for library amplification and target selection (Roche/NimbleGen SeqCap EZ Choice Library). A pre-capture amplification of the library was performed with ligation-mediated PCR (LM-PCR) using primers complementary to the adaptors, followed by two rounds of hybridization to the HPV-specific SeqCap EZ Choice Library. Amplified captured DNA from HPV infected cell lines, HeLa (HPV 18), CSCC7 (HPV16), and HPV TrDNA samples from premalignant cervical lesions were processed for multiplexed sequencing on the GS Junior system (Roche). Following completion of the pyrosequencing run, signal processing was performed, followed by detailed analyses. For the SYBR green qPCR assay we amplified an HPV E1 region common to thirteen high-risk HPV types for cervical cancer. Results: We isolated TrDNA from 10mL of urine samples. The mean concentration, total amount, and 260/280 ratio of isolated TrDNA was 34.2 ng/ul;1ug and 1.7 for premalignant samples and 17.2 ng/ul; 0.5ug; and 1.4 for normal samples. The total number of Pass Filter sequence reads was 100,428, the total number of bases over 41 million and the read length average was 130.3 bp. Most reads (91.63%) mapped to the reference HPV sequences, with the total number of mapped base pairs over 36 million (88%). The number of fully mapped reads was 14,899 (14.84%), and the number of partially mapped reads was 70,686 (88.17%). The average coverage of reference sequences was 78% for HPV 18 in the CSCC7 DNA, 86% for HPV 16 in HeLa DNA, as well as, 100% for HPV 18 and 96% for HPV 16 in TrDNA samples. The average Ct value for Pre-Capture LM-PCR DNAs was 35.74. The average Ct value for Post-Capture LM-PCR DNAs was 19.73; therefore the Delta Ct value was 16.01. Based on an estimated efficiency for the assay, the approximate fold enrichment was greater than 12,000. Conclusions: We demonstrated that the HPV genome can be assembled from HPV TrDNA. We are also the first to show that circulating high risk HPV TrDNA can be quantified using SYBR green qPCR assay after dual sequence capture of the HPV genome. Citation Format: Rafael E. Guerrero-Preston, Anne Jedlicka, Teresa Diaz-Montes, Josefina Romaguera, Juan C. Roa, David Sidransky. Identification of circulating HPV DNA in urine with a dual sequence capture approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-68. doi:10.1158/1538-7445.AM2013-LB-68

A Method for Metagenomics of Helicobacter pylori from Archived Formalin-Fixed Gastric Biopsies Permitting Longitudinal Studies of Carcinogenic Risk

The human microbiota has come into focus in the search for component causes of chronic diseases, such as gastrointestinal cancers. Presumably long induction periods and altered local environments after disease onset call for the development of methods for characterization of microorganisms colonizing the host decades before disease onset. Sequencing of microbial genomes in old formalin-fixed and paraffin-embedded (FFPE) gastrointestinal biopsies provides a means for such studies but is still challenging. Here we report a method based on laser capture micro-dissection and modified Roche 454 high-throughput pyrosequencing to obtain metagenomic profiles of Helicobacter pylori. We applied this method to two 15 year old FFPE biopsies from two patients. Frozen homogenized biopsies from the same gastroscopy sessions were also available for comparison after re-culture of H. pylori. For both patients, H. pylori DNA dissected from FFPE sections had ∼96.4% identity with culture DNA from the same patients, while only ∼92.5% identity with GenBank reference genomes, and with culture DNA from the other patient. About 82% and 60% of the predicted genes in the two genomes were captured by at least a single sequencing read. Along with sequences displaying high similarity to known H. pylori genes, novel and highly variant H. pylori sequences were identified in the FFPE sections by our physical enrichment approach, which would likely not have been detected by a sequence capture approach. The study demonstrates the feasibility of longitudinal metagenomic studies of H. pylori using decade-preserved FFPE biopsies.

A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species

Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.