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
Lenze: Their Sanger dropout rate is 10%; tested extraction (went from Q to Promega); library PCR got 3/4 products, good PGM data #IWT14
3:33am September 16th 2014 via Hootsuite
Lenze: Important points: amplifiability of DNA: they do a QC PCR for every sample, a four-plex test. Poor library PCR -> poor PGM seq #IW
3:32am September 16th 2014 via Hootsuite
Lenze: Now they apply 5% MAF minimum, 500x coverage of amplicons. 30 samples/week with one PGM. #IWT14
3:30am September 16th 2014 via Hootsuite
Lenze: Using titrated dilutions, mixtures got even to the lowest 1% MAF at 25x coverage. 'No false positives' #IWT14
Lenze: 49 comparison samples chosen, already Sanger sequenced. Also incl. complex deletion, all tested accurately on PGM #IWT14
3:29am September 16th 2014 via Hootsuite
Lenze: First it was 1 hotspot in 1 gene, now 6 hotspots in 2 genes. Ion PGM as suitable for FFPE due to low input, standard panels #IWT14
3:27am September 16th 2014 via Hootsuite
Next: Dido Lenze (Charité Pathology, Berlin) “Moving from Sanger to NGS” #IWT14
3:25am September 16th 2014 via Hootsuite
Trujillano: Upcoming work submitted in J Mol Diag, 75 prev. not described #IWT14
3:24am September 16th 2014 via Hootsuite
Trujillano: Since their pipeline was originally designed around WES, a different subset of tools used. #IWT14
3:16am September 16th 2014 via Hootsuite
Trujillano: Used Torrent Suite to obtain var calling and VCF output. They apply functional annotation and prioritization #IWT14
3:15am September 16th 2014 via Hootsuite
Trujillano: Another 95 samples with unk mutations were the challenge group. At 2000x depth, 99.99% of target at 20x (!) #IWT14
3:13am September 16th 2014 via Hootsuite
Trujillano: For hereditary breast and ovarian Ca; tested 150 samples previously sequenced on Sanger #IWT14
Trujillano: Decided to try AmpliSeq BRCA1/2 (see here http://t.co/QvbdgF7bnG ) Due to size of gene with Sanger, many missed var's #IWT14
3:10am September 16th 2014 via Hootsuite
Trujillano: Their unique CentoMD database; they est. only 50% of the deleterious var's are in the public domain #IWT14
3:08am September 16th 2014 via Hootsuite
Trujillano: WES at Centogene (CentoXome) is 120x, >95% at >20x, turnaround time is 1 mo. #IWT14
3:07am September 16th 2014 via Hootsuite
Trujillano: Emph of their Centogene is rare diseases. >350M people affected worldwide. #IWT14
3:05am September 16th 2014 via Hootsuite
Daniel Trujillano (Centogene AG, Rostock) NGS analysis of BRCA1 and BRCA2 genes #IWT14
3:04am September 16th 2014 via Hootsuite
Tyrelle:Q: Can pools of amplicons be analyzed as a group in Reporter? A: Can be done, if pool information is in the design file #IWT14
2:59am September 16th 2014 via Hootsuite
Tyrelle:Q: Use of informatic tools from other platforms? A:For annotation from VCF's, yes. Trio workflow requires variant caller, no #IWT14
2:57am September 16th 2014 via Hootsuite
Tyrelle: (HiQ con't): Still early with HiQ, will be of interest to see performance of HiQ on Proton #IWT14
2:56am September 16th 2014 via Hootsuite
