DT:Q:Diff between +/- selection? A:EPCAM RNA variable, and protein also not correlated well to RNA level. #AACR15
3:01pm April 18th 2015 via Hootsuite
DT: A: For human, flash freeze, of 500-600 whole transcriptomes, very similar results. Time is on the order of a few hours. #AACR15
2:55pm April 18th 2015 via Hootsuite
DT:Q:Cells in handling affecting RNA level? A:55/200 cells do not have RNA. Not only CTC problem though; timing is brief (3 min) #AACR15
DT: 2014 Cell ref http://t.co/TiIrx9SjSl with add'l ECM data. #AACR14
2:51pm April 18th 2015 via Hootsuite
DT: RNA in-situ - very important validation of their RNA-Seq data. Showed knockdown of SPARC. Other ECM proteins tissue-spec #AACR15
2:50pm April 18th 2015 via Hootsuite
DT: Comparing bulk primary vs single primary tumor - major loss of signal for ECM protein genes. SPARC RNA-ISH in mouse & human #AACR15
2:48pm April 18th 2015 via Hootsuite
DT: Showed majority of CTCs express Igfbp5 by RNA-ISH of primary tumor #AACR15
2:47pm April 18th 2015 via Hootsuite
DT: 7 pathways - MAPK, TGF-beta significant. 'CTC's have even more MAPK' than the primary tumor. #AACR15
2:44pm April 18th 2015 via Hootsuite
DT: Found 3 types of pancreatic CTC classes of genes, described in 2014 Cell Reports ref http://t.co/aKaDXZ8vV7 #AACR15
2:43pm April 18th 2015 via Hootsuite
DT: Due to high variability in transcriptome representation from single cells - high % of 'zero reads'; used rank product method #AACR15
2:41pm April 18th 2015 via Hootsuite
DT: Unsupervised clustering, primary tumors cluster together, showing 3 groups. Cell lines most homogeneous #AACR15
2:38pm April 18th 2015 via Hootsuite
DT: 3rd gen CTC-iChip has a single-file readout 2013 Sci Trans Med ref http://t.co/yw5nlTxA6P Used @Appliedbio 5500xl #AACR15
2:37pm April 18th 2015 via Hootsuite
DT: Many technologies; one review. http://t.co/3Lq4whktnQ ID'd Wnt2 in 2012 Nature but was EPCAM #AACR15
2:35pm April 18th 2015 via Hootsuite
DT: CTC need to intravasate (go into circ.), then extravasate (get out). CTC's implies heterogeneity. Liquid biopsy; localized ca #AACR15
2:32pm April 18th 2015 via Hootsuite
David Ting (Mass Gen Hospital MA) “Single cell RNA sequencing of circulating tumor cells” DT #AACR15
2:30pm April 18th 2015 via Hootsuite
CZ: They are working on exact linearity for WGA method; also low-level single-cell RNA-Seq, and single-cell ChIP-Seq. #AACR15
2:27pm April 18th 2015 via Hootsuite
CZ: First-strand error is 1e-4; thus one genome has 1e5 false-positives (!). Used 3 cells of very similar lineage to eliminate FP's #AACR15
2:22pm April 18th 2015 via Hootsuite
CZ: Method is not linear; called quasi-linear. Get 93% coverage at 25x depth. Cp to MDA (Genomiphi v2). Much better CNV resolution #AACR15
2:19pm April 18th 2015 via Hootsuite
CZ: Schematic from 2012 Science paper http://t.co/P4QwmDdrp0 explaining MALBAC. Sets of semiamplicons & full amplicons looped out #AACR1
2:17pm April 18th 2015 via Hootsuite
CZ: The Big Bang theory - 2015 Nature Gen ref http://t.co/2LWHw6mj6O argument for single-cells. Problem: Uniform amplification #AACR15
2:15pm April 18th 2015 via Hootsuite
Chuck Zong (Baylor, Houston TX) “MALBAC whole genome amplification of single cancer cells” #AACR15
2:13pm April 18th 2015 via Hootsuite
PVL: Single br ca cells: used Rubicon PicoPlex, MDA and PCR. Data from 2013 Nucl Acids Res ref http://t.co/EFiVopa9d3 #AACR15
1:58pm April 18th 2015 via Hootsuite
RT @DebGrainger: Prob. one of the most "out there" slides I've seen at a scientific meeting. Ever. Found at #AACR15 http://t.co/7FbecQWUV5
1:53pm April 18th 2015 via Hootsuite
PVL: Shows fig 2010 PNAS allele-spec copy number variation http://t.co/3wIb4eTKth TGP phasing, clear changes 'Battenberg' cake-like #AACR15
1:47pm April 18th 2015 via Hootsuite
PVL: 'Most recent common ancestor' cell, 2012 Yates ref Nature Gen Rev http://t.co/hlfUSQIKOf #AACR15
1:43pm April 18th 2015 via Hootsuite
PVL: SKY karyotypes shown; genomes are clearly v. aberrant. Nice figure from '13 Stratton EMBO ref http://t.co/l8LvnM42Hw #AACR15
1:41pm April 18th 2015 via Hootsuite
Peter Van Loo (Francis Crick Inst London) “Molecular archeology of cancer” PVL #AACR15
1:38pm April 18th 2015 via Hootsuite
NN:Q:Any animal model data? A:Only starting, no data yet. #AACR15
1:36pm April 18th 2015 via Hootsuite
RT @LAbizar: #AACR15 Navin, tumor evolution by single cell seq shows punctuated evolution http://t.co/F2J9N85LSu
RT @LAbizar: #AACR15 Navin, MD Anderson, future directions in single cell sequencing http://t.co/Xu1dSgwg2s
1:35pm April 18th 2015 via Hootsuite
NN:Q:How consistent? A:Var between pts much higher than within individual. SC - all common lineage #AACR15
1:34pm April 18th 2015 via Hootsuite
NN: Shows evidence for punctuated evolution fig from 2014 Nature http://t.co/gnxbLTSKvL Barcoding 'shows many subclonal muts at <1%' #AAC
1:32pm April 18th 2015 via Hootsuite
NN: Doing molecular barcoding and ultra-deep (118K-fold) seq, to sequence in bulk but validate single-cell exome seq Can see 1/5,000 #AACR15
1:30pm April 18th 2015 via Hootsuite
NN: From 16 cells from same individual: some substructure there, huge diff between tumor & normal. #AACR15
1:29pm April 18th 2015 via Hootsuite
NN: Sometimes - muts in normal cells, could be mosaicism in norm dev. Onto TNBC: ER-/PR-/Her2-, two major populations by CNV #AACR15
1:28pm April 18th 2015 via Hootsuite
NN: ER+ single-cell seq for br ca: showed CNV heatmap, exomes show much more diversity. PIK3CA in almost all; CASP3 about 80% #AACR15
1:26pm April 18th 2015 via Hootsuite
NN: Looking at tumor evolution: 2011 Nature http://t.co/gwuAnjGR7N Single nucleus sequencing 2015 Genome Biol http://t.co/D5KxUVZMhG #AACR15
1:25pm April 18th 2015 via Hootsuite
NN: 'Lots of commercial solutions' - Fluidigm's C1, Nanostring, SiliconBiosystems, Clontech, BD, QIAGEN listed #AACR15
1:22pm April 18th 2015 via Hootsuite
NN: Coverage uniformity 'still a big issue'. Validation is a need. Concern: microbial contamination. Very pure reagents needed. #AACR15
1:21pm April 18th 2015 via Hootsuite
NN:Errors: allelic dropout 10-50%; FP 1e-5 to 1e-6; FN coverage 10-50%. Tech variation / errors 'can be falsely interpreted' #AACR15
1:19pm April 18th 2015 via Hootsuite
NN: @iontorrent Proton shown, among other platforms. 'Emerging technologies' (single-molecule) can be useful for single microbes #AACR15
1:18pm April 18th 2015 via Hootsuite
NN: Whole transcriptome: oligo-dT anchor, template switching. New method: random index (http://t.co/oJmuO5c3HU) #AACR15
1:17pm April 18th 2015 via Hootsuite
NN: WGA: DOP-PCR is degen. primers, mostly used for copy-number. MALBAC mentioned. #AACR15
1:16pm April 18th 2015 via Hootsuite
NN: For rare cells (<1%): CellSearch, DEPArray, CellCelector (microcapillary), MagSweeper, Nanofilters (size-exclusion, 7um pores) #AACR1
1:14pm April 18th 2015 via Hootsuite
NN: Methods: isolating can be by mouth pipetting, serial dilution, flow-sorting (we use 'quite a lot'), microfluidics (C1), LCM. #AACR15
1:13pm April 18th 2015 via Hootsuite
NN: Mentions 2013 Nature Methods 'method of the year' issue as a nice overview (although not focused on cancer). #AACR15
1:12pm April 18th 2015 via Hootsuite
NN: Future directions - single cell epigenome, multiplexing, RNA & DNA from same cell. #AACR15
NN: 2014 review http://t.co/ehY6mI1JoR A diversity index can be calculated to guide therapy, predict response, monitor, early det. #AACR15
1:10pm April 18th 2015 via Hootsuite
NN: Publication numbers and distribution - most pubs are on cancer. Navin & Wang review Mol Cell in press #AACR15
1:08pm April 18th 2015 via Hootsuite
NN: Leeuwenhoek & Hook 1600's; Virchof's cell theory, considered 'father of pathology'. Karyotyping, FISH http://t.co/LCYOHvYvgt #AACR15
1:06pm April 18th 2015 via Hootsuite
