Sequencing

Recommendations

Info

11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting A FAST AND RELIABLE AMPLICON-BASED NGS STRATEGY FOR SCREENING OF GERMLINE AND SOMATIC MUTATIONS IN BRCA1 AND BRCA2 GENES Wednesday, 1st June 17:55 La Fonda Ballroom Tech Talk (TT‐1.05) Jiri Nehyba, Radmila Hrdlickova, Josh Kinman, Carrie Firmani, Dawn Obermoeller, April Lewis, Masoud Toloue Bioo Scientific Germline mutations in BRCA1 and BRCA2 genes are linked to hereditary breast and ovarian cancer. Individuals in affected families have heightened risk for breast and ovarian cancer and significant risk for prostate and pancreatic cancer. Somatic mutations in BRCA1 and BRCA2 also contribute to cancer development. Next generation sequencing (NGS) provides a convenient and simplified alternative to traditional Sanger sequencing to detect mutations in targeted BRCA genes. Sev‐ eral companies now offer BRCA NGS screening kits; however, there remains significant space for improvements in speed and cost efficiency. Bioo Scientific has developed five high performance amplicon assays for detection of BRCA1 and BRCA2 mutations. These assays differ in the type of samples for which the panel was optimized (either fresh/frozen or DNA isolated from FFPE samples), sequencing platform compatibility (Illumina or Ion Torrent), primer design, and in PCR‐based strategy for enrichment of the BRCA targets. Performance of these assays was evaluated for uniformity and depth of coverage, and reliability of mutation calling. Bioo Scientific’s assays were then directly compared to competitor assays using fresh/frozen DNA. FFPE samples of varying levels of degradation were tested using the NEXTflex BRCA1 and BRCA2 Amplicon Panels. All five Bioo Scientific assays for the detection of BRCA1 and BRCA2 germline and somatic mutations offer 100% target coverage, totaling 16.4 kb of the genome including all coding exons of both genes. Pathological mutations were correctly detected with these kits in six validated DNA samples from breast cancer patients (obtained from the HapMap project). In comparison with competitor kits, NEXTflex BRCA1 and BRCA2 Amplicon Panels can handle lower amounts of starting material with a shorter protocol for successful library preparation. Using two primer pools for fresh/frozen DNA samples enables the production of quality results with as little as 20 ng of DNA in only 4 hours. Library preparation for FFPE samples uses four pools, requires a minimum of 40 ng of input, and was tested to work with varying qualities of FFPE DNA preparations. Importantly, the NEXTflex BRCA1 and BRCA2 Amplicon Panels surpass other kits with regard to coverage uniformity and specificity, by reducing the amount of off‐target reads and by ensuring equal amplicon performance. Bioo Scientific’s assays provide >99% uniformity of amplicon reads for FFPE samples (100% for fresh/frozen DNA samples) and attain >97% specificity, defined as reads mapping within the desired target (>97.5% for fresh/frozen DNA samples). High uniformity and low off‐ target reads decrease sequencing costs, enabling a high level of multiplexing, especially when paired with NEXTflex 384 Illumina‐compatible barcodes. Because inherited and acquired mutations in BRCA1 and BRCA2 genes increase the risk of breast, ovarian, and several other types of cancer, the need to quickly and accurately detect these mutations is critical in research, preclinical, and clinical settings. The NEXTflex BRCA1 and BRCA2 Amplicon Panels are complete solutions, with high multiplexing capabilities, which bring efficiency, convenience, and speed to the detection of mutations associated with cancer in the BRCA1 and BRCA2 genes for several DNA sample types. 31
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting HIGH PERFORMANCE, STREAMLINED METHODS FOR RNA-SEQ AND TARGET ENRICHMENT Wednesday, 1st June 18:05 La Fonda Ballroom Tech Talk (TT‐1.06) Maryke Appel Kapa Biosystems Roche Diagnostics The expanding scope and application of next‐generation sequencing in both research and clinical environments have been driving a demand for sample preparation methods that yield high‐quality libraries, while supporting a higher degree of workflow automation and faster turnaround times. We have previously reported on advances in DNA library construction—most notably the incorporation of low‐bias enzymatic fragmentation in a streamlined, single‐tube workflow (KAPA HyperPlus). This method offers the speed and convenience of tagmentation‐based protocols, but consistently outperforms the latter with respect to library yields, key sequencing metrics (coverage uniformity and depth), and flexibility across different sample types and experimental designs. We have recently expanded our suite of streamlined sample preparation methods to include a complete, fully automatable workflow for target enrichment (HyperCap), as well as a novel, rapid workflow for the construction of stranded RNA‐Seq libraries (KAPA RNA Hyper Prep). The HyperCap workflow, co‐developed by Kapa Biosystems and Roche Nimblegen, incorporates the KAPA Hyper Prep or HyperPlus chemistry in an application‐specific approach to rapid target capture. General improvements to both the library construction and target capture portions of the workflow reduces turnaround time and eliminates steps that previously required user intervention and/or specialized equipment, and have therefore been difficult to automate. A series of optional improvements allows the end‐user to further tailor the protocol to specific sample types, sequencing applications and operational objectives. In its most extreme form, the HyperCap workflow allows for the construction of high‐quality, sequencing‐ready libraries from input DNA in ~9 hours. A more conservative, 2‐day workflow is recommend for challenging samples (e.g. low‐input FFPE) and/or small capture panels. Examples from of both ends of this spectrum will be presented. The KAPA RNA Hyper Prep workflow employs novel chemistries that allow for the combination of several steps in the construction of RNA‐Seq libraries. As a result, sequencing‐ready libraries can easily be prepared from total RNA in a standard 8‐hour day, inclusive of RNA enrichment (mRNA Capture or ribosomal depletion). The protocol is also compatible with total RNA input for RNA capture applications. Higher library construction efficiency allows for successful library construction from lower RNA inputs, and higher success rates with FFPE samples. Data generated with Universal Human Reference RNA (UHR), ERCC spike‐in controls and RNA isolated from fresh frozen and FFPE tissues will be presented. Products are for life science research use only, not for use in diagnostic procedures. 32
Page 1 and 2: Sequencing, Finishing, Analysis in
Page 3 and 4: 11th Annual Sequencing, Finishing,
Page 5 and 6: xGen ® Exome Research Panel • Re
Page 31: 11th Annual Sequencing, Finishing,
Page 83 and 84:
11th Annual Sequencing, Finishing,
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158:
Page 159 and 160:
Page 161 and 162:
Reliable solutions for focused NGS
Page 163 and 164:
Page 165 and 166:
Page 167:
166
show all

Sequencing

Create successful ePaper yourself

Delete template?

Save as template?