Impactpipeline is a perlpythonr commandline software toolkit to process data, call somatic aberrations snv,indels,sv,cnv generated by ngs based mskimpact assay. The performance characteristics of hcii assay and the pcr for detection of hpv dna have been compared in cervical lesions and results shown variation. In this project, a diagnostic custom hybrid capture based assay was tested on ffpe material to enable the simultaneous detection of all genetic alterations and to replace fish and ampliconbased parallel sequencing with one assay only. Comparative analysis of human papillomavirus detection by hybrid capture assay and routine cytologic screening to detect highgrade cervical lesions. Clinical validation in the context of a hybrid capturebased. The foundationfocus cdxbrca assay uses a customdeveloped analysis pipeline to identify brca12 base substitutions and short insertiondeletions indels up to bp. To test a new assay for the detection of human papillomavirus hpv dna, hybrid capture ii hc ii, compared with the previous commercialized hybrid capture i hc i and polymerase chain reaction pcr results on cervical scrapes from fresh cone excision biopsy samples. For exome captures, multiplexing has been tested on up to 12 samples 6 g total dna with limited impact on data quality. Nextera tagmentation coupled with optimized target capture ensures the fastest enrichment workflow time for custom content. The qiagen hc2 test is a sandwich capture molecular hybridization assay. Assessment of capture and ampliconbased approaches for. Hybrid capture provides superior assay sensitivity compared with amplicon sequencing. Ville jussila, and julia ruotsi for assistance with storing data into spss files.
Amplificationbased methods have excellent specificity and uniformity. Class of 2018 get together to utilize active learning and show our take on what exactly hybrid. Hybrid capture and nextgeneration sequencing identify. Enrichment capture methods for ngs are widely used, however, they evolve rapidly and it is necessary to periodically measure their strengths and weaknesses before transfer to diagnostic services. Analysis workflow for development of capture based targeted sequencing assay, progressing from platform selection to the reporting of mutations. Hpv typing by the dna assay can be performed either from a cervicovaginal sample collected into a. The foundationfocus cdxbrca assay uses a customdeveloped analysis pipeline to identify brca12 base substitutions and insertiondeletions indels. The goal of this project was to design and test a probe capture assay targeting forensically relevant nuclear dna snps for mps of degraded and limited dna samples as well as mixed dna samples. The assay is called hybrid capture hc whole genome amplification wgaluminex lmx and is described as follows. Hybrid capture article about hybrid capture by the free.
A secondgeneration commercial hpv test, the hybrid capture 2 hc2 assay, was. These keywords were added by machine and not by the authors. Such data will permit objective interpretation of this new procedure, and could contribute to cancer prevention. Herein, we present the clinical validation of 76 genes from a novel 1212gene largescale hybrid capture cancer sequencing assay university of chicago medicine. Clinical validation of a nextgeneration sequencing genomic. We evaluated the performance of the hybrid capture 2 ctgc hc2 assay with the digene rapid capture. Both methods proved clinically useful for detecting patients at risk of developing cmv disease and for. The performance characteristics of pcr and the hc2 assay for testing for hpv have been compared, and the results have varied 2, 10, 14, 19, 24, 26. The hybrid capture 2 hc2 system hpv dna test by digene by brenda palacios objectives state the 2 main lowrisk hpv dna types state the 2 main highrisk hpv dna types state what type of test method is used for the detection of hpv state the minimum sample volume required for testing state what the molecular sandwich consist of human papillomavirus hpv primary etiological agent in cervical. Hybrid capture 2 hc2 hpv dna test qiagen, a nucleic acid hybridization microplate assay with signal amplification that utilizes chemiluminescent detection. In order to investigate the reliability of detecting hpv dna in cervical smears, we compared the performance of nested mygp pcr and fda approved hybrid capture ii hcii using clinical cervical scrapings from 40 patients.
Hybridcapture based sequencing assays to detect novel. The rapid rate of adoption has been facilitated by falling reagent costs, benchtop instruments, improved chemistries and improved data analysis solutions. Refer to the respective manufacturers package inserts for instructions for using preservcyt solution for collection, transport, storage, and preparation of specimens for use in those systems. Research open access comparative study between the. Roche genotyping assay was used to resolve discordant hc2 and. First, there is a sequencespecific sample enrichment process to isolate singlestranded hpv dnas while removing much of the nonspecific genomic dna that may interfere with some assays. Download acrobat pdf file 61kb supplemental figure s2. The bdna method was initially developed by chiron emeryville, ca, usa and marketed by bayer diagnostics emeryville, ca, usa, and the hybrid capture method was developed and marketed by digene corporation gaithersburg, md, usa. The probes are solutionbased, biotinylated oligonucleotide sequences that are designed to hybridize and capture the regions intended in the design. Pdf evaluation of the digene hybrid capture ii assay. Targeted enrichment by solutionbased hybrid capture to. The detection of the pcr products was performed by the eia procedure, as described. Hybrid capture is a signal amplification technique, meaning that the chemiluminescent or fluorescent signal is amplified to aid detection, rather than the target dna being amplified by pcr. Industry experts, and our own experts reorganized and improved the content so that you find the trusted answers that you need for genomics products like.
A comparison of the roche cobas hpv test with the hybrid. These values are downloaded to an excel file for data storage and subsequent file transport to westat. Transitioning from custom ampliconbased parallel sequencing. The hc2 highrisk hpv dna test procedure, quality control, and the. Detection of human papillomavirus dna by the hybrid capture assay. Lab medicine a proprietary system used to detect and monitor viraleg, chlamydia spp, cmv, hbv, hpv infections. The hybrid capture ii test is a nucleic acid hybridization assay where. In addition, printed copies of each assay, together with quality control calculations, are stored in notebooks.
Specimens containing the target dna hybridize with the high risk hpv rna probe cocktail whole genomic probes for high risk hpv. Pdf clinical relevance of the borderline results of the hybrid. Hybrid capturebased enrichment methods use sequencespecific capture probes that are complementary to specific regions of interest in the genome. Comparison of onclarity human papillomavirus hpv assay.
Elisa enzymelinked immunosorbent assay is a platebased assay technique designed for detecting and quantifying peptides, proteins, antibodies and hormones. Comparison with hybrid capture i and pcr results in cervical lesions. Molecular diagnostics of human papillomavirus laboratory. This elisa technical guide will increase your awareness of elisa techniques and help you maintain proficiency with this methodology. Commonly used signal amplification technologies include branched dna bdna and hybrid capture hc assays. To compare the performance of the roche cobas and hybrid capture 2 tests for the detection of high. Input for capture we recommend using 500 ng of each prepared library for hybrid capture. Dec, 2014 first year medical students from philadelphila college of osteopathic medicine georgia campus d. The aim was to translate the new assay into clinical practice to advance the establishment of targeted therapies. Comparison of analytical and clinical performance of clart. Streamengine, camengine, or other related software and firmware can be downloaded and installed through assist central. The dish is peppered with small, single stranded dna molecules or chains that stick up into the air.
Hybrid capture ii, a new sensitive test for human papillomavirus. Selecting the best ngs enrichment assay for your needs introduction next generation sequencing ngs is now in routine use for a broad range of research and clinical applications. In the nuclease hybridization assay, the oligonucleotide analyte is captured onto the solid support such as a 96well plate via a fully complementary cutting probe. Hybrid capture assay, version hc2 digene, gaithersburg, md, usa which is able to detect highrisk. Briefly, the capture probes were assayed for biotin incorporation by binding 500 ng of biotinylated pcr products to 200. Next generation sequencing for solid tumors diagnostics. Comparative study between the hybrid capture ii test and. Hybrid capture assay definition of hybrid capture assay by. Human papillomavirus testing with the hybrid capture 2 assay and. On september 29th, agilent launched a major web update for our line of genomics products. Successful hybrid capturebased ngs is influenced by a range of parameters including probe design efficiency, ontarget coverage, uniformity of coverage, analytical sensitivity and specificity, amount of dna input required, scalability, reproducibility, ease of use, and overall cost 1, 2, 3. Chlamydia trachomatis capture probe hybrid capture digene hybrid capture hybrid capture method.
Comparative study between the hybrid capture ii test and pcr. Rlu values are captured by assayspecific software on a dedicated computer attached to the luminometer. Hpv genotype detection using hybrid capture sample. Crossreactivity profiles of hybrid capture ii, cobas, and.
Organization and format errata, author corrections, retractions illustrations and. Thednarnahybridsarecapturedon amicroplate,andtheemittedlightismeasuredinaluminometerasrela. It demonstrates superiority of hybrid capture sequencing over ampliconbased method with respect to assay. To meet the increasing demand for testing, a highthroughput system is desirable. Comparison between the hybrid capture ii test and a pcrbased. Recentaly, a secondgeneration assay with improved diagnostic sensitivity has been developed known as hybrid capture ii test hcii and approved by the us food and drug administration fda.
Crossreactivity has only been systematically and independently evaluated for the most widely used assay, hybrid capture ii hc2, where it was most frequently due. The capture assay was designed to capture 600 kb of target sequence which corresponded to 1. Comparison between the hybrid capture ii test and a pcr. Analytical validation of a hybrid capture based nextgeneration sequencing clinical assay for genomic profiling of cellfree circulating tumor dna. Specimens containing the target dna hybridize with a specific hpv rna probe cocktail. While wes has proven useful for diagnosis and management in specific clinical situations, selected gene panels have more utility at lower cost. Human papillomavirus testing with the hybrid capture 2 assay. Cervical cancer due to human papillomavirus hpv is. A recent publication reports a protein a capture lcmsms hybrid assay with papain a cysteine protease cleavage for the quantitation of valinecitrullinlinked mmae adcs 34. We evaluated the performance of the hybrid capture 2 ctgc hc2 assay with the digene rapid capture system hc2rcs. Pcr products of the appropriate size were then gel purified, and an aliquot of each 250 to 500 ng was subjected to a bind and boil assay to determine the extent of biotin incorporation.
Comparison of onclarity human papillomavirus hpv assay with hybrid capture ii hpv dna assay for detection of cervical intraepithelial neoplasia grade 2 and 3 lesions. Detection of human papillomavirus dna by the hybrid capture assay maria odete o. Quantification of cytomegalovirus cmv viral load by the hybrid capture assay allows for early detection of cmv disease in lung transplant recipients. This document details the lab protocol for nhanes 2003. Introduction pathogens often cause infectious diseases and significant. The test provides a qualitative molecular detection of different human papillomavirus highrisk types. The assay is using s1 nuclease, which degrades singlestranded dna and rna into oligo or mononucleotides, leaving intact doublestranded dna and rna.
Human papillomavirus testing with the hybrid capture 2. Enzymelinked immunosorbent assay elisa is an antibodybased method indirect elisa sandwich elisa elispot competitive elisa direct elisa designed to quantitatively or quantitatively detect a specific antigen proteins, peptides, hormones or an antibody in a sample sample types. The digene hpv test using hybrid capture ii technology is a nucleic acid hybridization microplate assay with signal amplification that utilizes chemiluminescent detection. The hybrid capture assay is a standardized assay that is simple to perform and can utilize whole blood specimens that have been stored for up to 48 h. This study focusing on the development of a sequencing pipeline to personalize lymphoma management establishes feasibility of capture sequencing of routinely acquired ffpe tissue dlbcl and fresh cell preparations cll in a single assay. This process is experimental and the keywords may be updated as the learning algorithm improves. As with any assay, the reproducibility and reliability of elisas depend upon proper technique and attention to detail. Detection of human papillomavirus dna by the hybrid. Aptima hpv assay panther system 100 and 250 test tigris dts system 250 test. The seqcap probes are solutionbased, designed to be specific for the target regions of interest and ready to hybridize to a sequencing library in order to enrich for targets of interest. The flexible, fully customizable design accommodates up.
It is designed for use with hybrid capture, including both wholeexome and custom target panels, and shortread sequencing platforms such as illumina. Pdf analytical validation of a hybrid capturebased next. In an elisa, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Step 3 test for selfactivation measure his3 basal expression due to selfactivation in both strains pdbleux and ppc86y increase 3at in yeast media plates w histidine 3at inhibits his3 in dosedependent manner titrate his3 activity until growth is inhibited determine minimum effective dose allows for weak proteinprotein interactions to be detected. Aptima hpv e6e7 mrna test is as sensitive as hybrid capture 2 assay but more specific at detecting cervical precancer and cancer. Briefly, hc2 is a signal amplification assay that uses a combination of antibody capture and chemiluminescent signal detection. Panel design, hybrid capture, and sequencing solution hybridization was performed using a 50fold molar excess of a pool of 2695 individually synthesized 120bp 50biotinylated singlestranded dna oligonucleotide baits assay baitset version cf2. The flexible, fully customizable design accommodates up to 15 mb of. Methods for detection of hpv infection and its clinical utility iarc. This document details the lab protocol for nhanes 20032004. Pdf detection of human papillomavirus by hybrid capture and real. Hybrid capture 2 detected lr hpv for 15 specimens, 12 of which were confirmed. Analytical validation of a hybrid capturebased next. Get a printable copy pdf file of the complete article 680k, or click on a page.
Cytological diagnoses were reported using the 2001 bethesda system14. Ngs assay development ngs allows many types of anomalies in many genes simultaneously. Method development and assay validations for the analysis of. Pdf the hybrid capture 2 hc2 highrisk hpv dna assay serves as a triage test in the slovenian national cervical cancer screening programme zora.
Using less input for capture can result in higher duplicate rates, lower mean coverage, and poor coverage uniformity. This assay aids in the diagnosis of sexuallytransmitted hpv infection and is used for the triage of patients with an ascus pap test result. The original hybrid capture assay hc1 was approved by the fda in 1995. Determination of hpv dna viral load by hybrid capture assay and its. To test a new assay for the detection of human papillomavirus hpv dna, hybrid capture ii hc ii, compared with the previous commercialized hybrid capture i hc i and polymerase chain reaction. Furthermore, this microfluidic device has great potential in rapid detection of a wide variety of different other bacterial and viral pathogens. If thinprep vials are qns for the hybrid capture assay, then a test requiring a lower input volume will be automatically performed if there is sufficient volume to. Assessment of the latest ngs enrichment capture methods in. Comparison of the hybrid capture 2 and cobas 4800 tests for. Updates and information from rex healthcare and rex outreach. Teixeira, biomedical institute of universidade federal. Human papillomavirus detection by the hybrid capture 2 hc2. Collected vaginal swabs in rural uganda for detection of human papillomavirus.
Exclusively available for the live gamer and live streamer series. The assay is called hybrid capture hcwhole genome amplification wgaluminex lmx and is described as follows. Hpv vaginal swab digene high risk laboratory procedure manual. Foundationfocus cdxbrca technical information summary. Assessment of capture and ampliconbased approaches for the. We compared two common hpv dna detection tests hybrid capture ii hcii and pcrenzyme immunosorbent assay eia using the primers gp5 gp6 followed by hpv typing with reverse dot blot hybridization for sensitivity and speci. Pdf evaluation of the digene hybrid capture ii assay with. Check your product insert for specific instructions for each assay you perform. The hc2 highrisk hpv dna test using hybrid capture2 hc2 technology is a nucleic acid hybridization assay with signal amplification using microplate chemiluminescence for the qualitative detection of thirteen highrisk types of human papillomavirus hpv dna in cervical specimens. Hybrid capture definition of hybrid capture by medical.
Comparison of onclarity human papillomavirus hpv assay with. Pdf quantification of cytomegalovirus cmv viral load. Nextera rapid capture custom enrichment is an allinone assay for library preparation and custom target enrichment. Selecting the best ngs enrichment assay for your needs. A secondgeneration commercial hpv test, the hybrid capture 2 hc2 assay, was introduced as a readytouse test for routine diagnostics and includes negative controls and positive controls pcs. Many assay type choices with different pros and cons. Pdf evaluation of polymerase chain reaction pcr method. The hc2 highrisk hpv dna test using hybrid capture 2 hc2 technology is a nucleic acid hybridization assay with signal amplification using microplate chemiluminescence for the qualitative detection of thirteen highrisk types of human papillomavirus hpv dna in cervical specimens. Determination of cytomegalovirus dna load for monitoring. A branched dna assay begins with a dish or some other solid support e. Sep 23, 2010 recentaly, a secondgeneration assay with improved diagnostic sensitivity has been developed known as hybrid capture ii test hcii and approved by the us food and drug administration fda. Hybrid capture assay definition of hybrid capture assay. Comparative study between the hybrid capture ii test and pcr based assay for the detection of human papillomavirus dna in oral submucous fibrosis and oral squamous cell carcinoma ajay kumar chaudhary1,2, shruti pandya2, ravi mehrotra2, alok c bharti4, mangal singh3, mamta singh2 abstract background. Similarly, with the hybrid capture assay, the amount of cmv dna decreased about 100fold in five patients and decreased by about 0.