Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans , M . ozzardi , M . rodhaini and M . streptocerca . Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M . perstans and M . ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M . perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000 th of a microfilaria, while the M . ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M . perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.
Early detection and diagnosis of cancer substantially increases the likelihood for successful treatment. Tools that aid in detecting and diagnosing cancer early, therefore, have the potential to greatly impact the clinical outcome for cancer patients. Next Generation Sequencing (NGS) has emerged as an important tool in this area. The technology is sensitive, fast and high throughput to allow sequencing of many samples at once. Unfortunately, many clinical samples go unanalyzed because they do not yield sufficient quantities of DNA to generate NGS libraries or the libraries generated require so many rounds of PCR amplification that they display extreme sequence bias. Bias not only hampers data analysis, but also increases costs by requiring excess sequencing to obtain sufficient coverage over all relevant genomic regions. To enable the increased use of NGS in the clinic and reduce the amount of sequence bias generated during library preparation, we have developed a PCR free library construction method that uses low quantities of DNA as input. As an initial test of the method, we generated PCR free libraries from 100ng, 50ng and 25ng of human genomic DNA. The libraries where pooled and sequenced on the Illumina NextSeq 500 instrument to approximately 10X coverage. All libraries, irrespective of input amount, showed minimal AT/GC bias and excellent coverage distributions, with most bases covered within 5X of the expected coverage depth. In addition, regions identified as difficult to sequence (Aird, D., et.al., 2011 and Ross, M. G., et.al., 2013) showed coverage at near expected levels for all libraries. This method can easily be adapted for use with extremely low DNA inputs by the introduction of a minimal number of PCR cycles. In fact, we have used this method to construct high quality NGS libraries with picogram quantities of DNA input. Standard library construction methods require DNA inputs of 2ug to 500ng when PCR amplification is omitted. This new method utilizes inputs as low as 25ng to generate high-quality PCR free libraries and picogram quantities when amplification is performed. We are currently investigating the possibility of reducing input levels further and exploring the limits of the method with low quality DNA samples. Interestingly, we have observed substantial sample loss during DNA shearing and reaction cleanup. Samples that do not require fragmentation, such as DNA isolated from plasma (cfDNA) and low quality FFPE DNA, may reduce the input requirements even further. Finally, this new method utilizes low sample and reagent volumes, possibly paving the way for its use in microfluidic devices. Citation Format: Lynne Apone, Pingfang Liu, Vaish Panchapakesa, Deyra Rodriguez, Karen Duggan, Krishnan Keerthana, Nicole Nichols, Yanxia Bei, Julie Menin, Brad Langhorst, Christine Sumner, Christine Chater, Joanna Bybee, Laurie Mazzola, Danielle Rivizzigno, Fiona Stewart, Eileen Dimalanta, Theodore Davis. Enhancing clinical utility of NGS with reduced bias, low DNA input, library construction. [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 3620.
Next generation sequencing technology is quickly becoming the most rapid and accurate method of determining mutation profiles of patient's tumor samples. While whole genome or exome sequencing profiles are the most comprehensive methods for complete exploration of these tumors, these protocols typically require large amounts of DNA, are expensive, don't get deep coverage, and require extensive time for library and sample preparation. Ion Torrent has developed new methodology for looking at 10’s to 1000’s of genomic targets with the Ion AmpliSeq™ technology. The recommended input of 10ng of DNA is suitable for work with FFPE or fine needle biopsy samples, and has been demonstrated on samples with less than 1ng of DNA. In a 10 hour day this protocol can take isolated DNA through the library and sample prep as well sequencing with variant calls, making it an incredibly rapid method for determining sequence variants within targeted regions. There are currently two commercially available cancer-specific amplicon panels: the Comprehensive Cancer Panel (CCP) uses ∼16,000 primer pairs covering the exons of 409 tumor suppressor genes and oncogenes frequently cited and mutated in cancers, and the Cancer Hotspot Panel v2 (CHP2) uses 207 primer pairs in 50 clinically actionable genes to detect over 2800 mutation hotspots. We recently measured the sensitivity of SNP detection levels with the CCP and CHP2 panels on a 90:10 (reference:SNP) admixed sample and were able to detect SNP proportions of 2.0%(p<0.005) with CHP2 and 2.0% (p<0.001) with CCP. The CHP2 amplicons yielded coverages of >2000X for a single sample on a 314 chip and >3000X for 6 barcoded samples on a 318 chip. Comparatively, the average base coverage of the CCP was >280x and over 95% of the CCP amplicons were covered at >50X. We ran the CCP in quadruplicate on 318 chips at two separate facilities and the CHP2 was run barcoded on 318 chips and single samples on 314 chips also in quadruplicate at two separate facilities and all of the chips show very equivalent numbers for SNP detection and coverages. These results demonstrate the consistent nature of targeted sequencing using a next gen platform. Going from isolated DNA to variant calling in as little as 10 hrs, this method is extremely fast and efficient which will be critical in future diagnostic settings. Citation Format: Devin Dressman, Stefanie Nishimura, Charles Scafe, Yutao Fu, Ellen Fanti, Lev Kotler, Vaish Panchapakesa, Wendell Orphe, Sherry Hansen, Nick Hapshe, Carl Fergus, Erin Lagier, Eric Wei, Grace Lui, Reddy Nallapareddy, Xiaohui Chen, Zhitong Liu, Eugeni Namsaraev. Rapid, inexpensive and accurate detection of rare variants by targeted sequencing. [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 4215. doi:10.1158/1538-7445.AM2013-4215
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