Sartori: 3&4 modified conditions for G/C-rich templates; after amplifying combine 1&3, 2&4 for library prep. 'Everyone was nervo
8:50am September 16th 2014 via Hootsuite
Sartori: Without a new design, they added two more pools to the first 234 amplicons. 3&4 have only 12 & 15 primer pairs #IWT14
8:49am September 16th 2014 via Hootsuite
Sartori: 2 panel pool had low performance including CEBPA, DNMT3A. Many in the list were along CEBPA gene #IWT14
8:47am September 16th 2014 via Hootsuite
Sartori: For AML, collaborators from UCL (London), CHRU (Lille, France), CHUV (Lausanne) #IWT14
8:42am September 16th 2014 via Hootsuite
Sartori: Consortium proposed list, tested performance, verified with known samples, demo'd workflow #IWT14
8:41am September 16th 2014 via Hootsuite
Sartori: FALCON organizes consortia around applications; helps with analytical verification; tailor workflow dev't; share experience #IWT14
8:36am September 16th 2014 via Hootsuite
Sartori: Melanie Baumann from the FALCON team, a clinical consortium development facilitation group. Lab in Darmstadt #IWT14
8:34am September 16th 2014 via Hootsuite
Alexander Sartori (Thermo Fisher Scientific) "Ion AmpliSeq AML Research Panel" #IWT14
8:31am September 16th 2014 via Hootsuite
Gunther: Project: + / - cells, 50-200K cells (proliferated). r around 0.88-0.94. FACS takes time, primary culture affect r values #IWT14
8:22am September 16th 2014 via Hootsuite
Gunther: (cont) SAMTools, feature Counts, mergeCounts, correlation, DESEQ2 (more relaxed) for genes / isoforms) #IWT14
8:20am September 16th 2014 via Hootsuite
Gunther: Their bifx workflow: FASTQ, add’l quality trimming by Reaper (EBI Q20/20, 30-200bp), use STAR (Encode) for mapping... #IWT14
Gunther: Size-selected vs non-SS samples not to be mixed; less mappable reads. Readlengths >20-30% longer, better for de novo ass’y. #IWT
8:19am September 16th 2014 via Hootsuite
Gunther: Small input results in high duplications; reduce # of cycles cut dupl. rate from 60-70% to below 40%. Fewer PCR dup's in IGV #IWT14
8:18am September 16th 2014 via Hootsuite
Gunther: Changes to protocol: RNA frag to 1 min for up to 4ug starting amt. Need to aliquot ligation buffer (as the ATP is labile). #IWT14
Gunther: Tested sample preps for small RNA: Best results are PureLink miRNA; Q RNeasy Micro + on-column DNase #IWT14
8:17am September 16th 2014 via Hootsuite
Gunther: FACS muscle stem cells: Min input 10-20K cells (100ng total RNA input, more is better) #IWT14
8:16am September 16th 2014 via Hootsuite
Gunther: Proton, 2/3 of almost 100 runs RNA-Seq or miRNA-Seq. Use RiboMinus Eukaryote v2, also Dynabeads mRNA Direct Micro (polyA) #IWT14
8:15am September 16th 2014 via Hootsuite
Dr. Stephan Gunther (MPI Bad Nauheim) "Transcriptome analysis on Ion Proton™ Sequencer" #IWT14
8:11am September 16th 2014 via Hootsuite
Kurth: (Gave credit to the OncoNetwork Consortium, and to Rosella Petraroli of Thermo Fisher Scientific) #IWT14
6:41am September 16th 2014 via Hootsuite
Kurth: RNA fusion: 20k reads/sample, 16/318 chip, fusion positive if >20 reads across junction. Sens 97% by imbalance or by fusion #IWT14
6:36am September 16th 2014 via Hootsuite
Kurth: Serial dilution: fusion detection down to 1% diluted into HBR. >200 prev. tested samples. #IWT14
6:35am September 16th 2014 via Hootsuite
Kurth: Imbalance can pick up fusions where the partner primer is absent. Nice chart showing ALK pos to ALK neg as a fn of the assay. #IWT14
6:33am September 16th 2014 via Hootsuite
Kurth: Also: 3’-5’ imbalance assay, wild-type RNA have both 3’ and 5’ regions, fusions have only the 3’. #IWT14
Kurth: One AmpliSeq RNA fusion control is the housekeeping genes MYC, ITGB7, LMNA, HMBS, TBP #IWT14
6:31am September 16th 2014 via Hootsuite
Kurth: Laid all all fusion partners: ALK: 5; ROS1: 8; RET:3; NTRK1: 9. Includes pharma, Compendia, and COSMIC ones. #IWT14
6:29am September 16th 2014 via Hootsuite
Kurth: Lung Fusion AmpliSeq RNA: ASK / ROS / RET 3-8% occurrence in NSCLC: break-apart FISH not so happy with the handling of sample. #IWT14
6:25am September 16th 2014 via Hootsuite
Kurth: n=135 against http://t.co/rRAc0cVHl2: split across lung tumor frequencies pie chart almost identical. #IWT14
Kurth: JSI SW can archive all must including low freq (2 or 3%). Can help with artifacts or real mutations that can be annotated. #IWT14
Kurth: 2-45 samples / week. They use JSI medical systems. PE NGS Express (>8 samples) “Works very well”, also use Equalizer kit #IWT14
6:24am September 16th 2014 via Hootsuite
Kurth: With an n=116; add’l RAS in 11% discovered due to the new coverage. #IWT14
6:19am September 16th 2014 via Hootsuite
Kurth: They are a core facility for their different business units. Part of OncoNetwork consortium. Colon & Lung panel development #IWT1
6:18am September 16th 2014 via Hootsuite
Kurth: ‘How to become a fast reader’. Train the eyes, angle of the book, impt to read a lot. They decided to become fast DNA readers! #IWT14
Henriette Kurth (Viollier, Basel): “New tools for the genetic analysis of solid tumors” #IWT14
6:17am September 16th 2014 via Hootsuite
Bräuninger: Found NFKb activator in germline bone-marrow #IWT14
4:02am September 16th 2014 via Hootsuite
Bräuninger: Hemophagocytic syndrome I: rare immune system disorder, eating own blood cells. High mortality #IWT14
3:59am September 16th 2014 via Hootsuite
Bräuninger: Data from four lesions suggest all completely diff mutations. All independent mutations. Pub.: http://t.co/DZovnP2XIm #IWT14
3:57am September 16th 2014 via Hootsuite
Bräuninger: Second ex.: multifocal malignomas (multiple tumors in many sites). Colorectal adenocarcinoma, but simultaneous to lung #IWT14
3:55am September 16th 2014 via Hootsuite
Bräuninger: No affect on proliferation, but up regulated apoptosis. Kunze et al Cancer Res in press #IWT14
3:53am September 16th 2014 via Hootsuite
Bräuninger: PLCG1 is downstream EGFR, RAS-RAF and PI3K are well known; PLCG1 as activated signalling is novel #IWT14
Bräuninger: Final analysis, 15 coding muts; 11 genes, either signaling (ie. PLCG11), chromatin modifier (MLL2), Other #IWT14
3:51am September 16th 2014 via Hootsuite
Bräuninger: Of 18, 6 had enough for matched pairs to analyze. Success of all 12 (6 T/N pairs), 128 SNV, 20 indels #IWT14
3:50am September 16th 2014 via Hootsuite
Bräuninger: Primary cardiac sarcomas rare; no completely excision possible, chemo- and rad-resistant. 18 samples in 10y #IWT14
3:49am September 16th 2014 via Hootsuite
Bräuninger: Want to do WES or WGS, but limiting material is a problem. CCP (compreh. cancer panel) has 409 genes #IWT14
3:48am September 16th 2014 via Hootsuite
Prof Andreas Bräuninger, Justus-Liebig-University Gießen: Detection of mutations with the Comprehensive Cancer Panel in Clinical Res. #IWT14
3:47am September 16th 2014 via Hootsuite
Lenze:Q:How many Sanger verified? A: "Almost none" - due to quality of data, it's the calling and annotation #IWT14
3:43am September 16th 2014 via Hootsuite
Lenze: Summarize: Promega Maxwell extration plus Zymo columns; Ion Chef purchased but not tested yet for workflow automation #IWT14
3:41am September 16th 2014 via Hootsuite
Lenze: If no variants called - to inspect quality metrics; Use of Sample ID set they find very helpful. #IWT14 http://t.co/nwIB4nQvLV
3:39am September 16th 2014 via Hootsuite
Lenze: Most confident with Torrent variant caller + IGV. Manual inspection. For complex indels (say with KIT) - look for homopolymers #IWT14
3:38am September 16th 2014 via Hootsuite
Lenze: Workflow complexity: AmpliSeq # PCR cycles can vary; need for accurate lib quantitation; consistent loading needed #IWT14
3:36am September 16th 2014 via Hootsuite
Lenze: Tested AMPure beads, found Zymo PCR inh. removal also helpful. Success rates with PGM now comparable to Sanger (~90%) #IWT14
3:34am September 16th 2014 via Hootsuite
