Friday 05/17/15

9:00 am – 5:00 pm

9:00 am – 3:00 pm

  • review and prep slides for group meeting journal club
  • lab meeting (see notes) 10am – 1pm
  • reformatting figures for Ph paper

Transcriptome design

  • might have been faster to use cufflinks gtfread to output fasta for all transcripts, then to cut on this, rather than parse with my own matlab code.
  • this could be made into a par for loop if I managed to get rid of the eval statements (which are dealing with colons)
  • as stands assembly should be finished by Monday
  • moving ahead with 16 genes.

Doory Defense 3:00 pm – 4:00 pm

Discussions

  • possible meeting with Ting
  • discussion with Ting about stuff
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Thursday 04/09/15

9:00 am – 8:00 pm

Ph project

Figure making

  • revising layout of Fig 5
  • sent to team
  • NF says a bit too tight, need more whitespace.
  • re-arranged panels again

MERFISH

  • exploring probe selection criteria
  • compare variable length probes to 20mers with 20% GC range (35-55%)
    comp20mers

Building better transcriptome

  • approach: combining ENCODE data from multiple cell lines to get a broader swath of genes that are appreciably on.
  • selected cell lines: ‘a549′,’daoy’,’hct116′,’imr90′,’mcf7′,’panc1′,’t47d’,’u2os’
  • require gene to be at FPKM > 1E-5 in one of these cell lines.
  • challenge 1: need to chose an isoform to build probes to
  • challenge: isoforms rather cell specific:

FLNA MYH10

  • current solution: select isoform with the highest average expression across all cell lines.
  • new human transcriptome representing expression FPKM >10^-5 now running
  • note 20K of these 45K genes has FPKM > 1 in one of these cell lines.
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Wednesday, 04/15/15

9:00 am – 10:00 pm

MERFISH

Code development

  • discuss Jeff’s new approach to Library construction
  • Fix indexing in Assemble Oligo Array probes.
    • complete. see Library11CompileOligoArrayResults_150414.m
  • BLAST combos of primers and new Fast Secondaries
    • completed for 5′ fusions. killed half of ~500 remaining secondaries.
    • now doing for 5′ and 3′ rev-complement primer fusions.
  • library 12 passed checks finally! Sent to Jeff and team
  • lib12 sent to CustomArray, requested PO

Ph project

  • revising text
  • writing model description
  • writing figure caption
  • adjusting figure
  • sent to NF and AW
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Tuesday 04/14/15

10:00 am – 8:20 pm, 9:20pm – 12:00am

MERFISH

  • reply to Quake lab with raw data
  • working on fixing lib12 build
  • found bug in my matlab BLAST — allhits field not being populated if no varargin’s passed. fixed.
  • updating MERFISH team on discussions with collaborators, 12:00pm-12:40pm
  • adjusting Library building script to allow buffering of total probe numbers 1:30pm – 3:00pm
  • need to shuffle pri-secondary combos and secondaries when changing to new secondaries. (primary encoding sequences will be the same).
  • running script to assemble library 12, 3:00pm – 4:00pm

adding remove probes so as to balance bits 4:00pm – 7:00 pm

  • better to do based on bit combos
  • potential bug in probe assembly: probe names are non-unique. Not certain targeting sequences selected at random are being selected without replacement… Need to investigate this.
  • Investigated: these are from different 1kb sections, that happen by chance to have the same base end. Hence our sequence IDs are non-unique. should fix this for ID purposes, but makes no difference for probes.

Running probe building (again), 7:00pm – 12:00am

  • serious problems with FAST libraries losing whole bits or large number of bits when fused to primers in the final BLAST.
  • wrote loop to run through primers if we don’t keep at least 75% of the least abundant bit combo (since everyone will be pruned down to the least abundant).
  • FAST secondaries + lib5 primers are getting destroyed by BLAST to abundant genes and rRNA/tRNA.
  • memory is getting destroyed by BLAST
  • launching on Morgan, 10:15 pm
  • Writing to TSTORMdata2 is at least 10x slower than writing straight to disk, even from Morgan. Nope. 10x was too optimistic. Monet lapped Morgan in printing libraries. TSTORMdata2 is probably closer to 100x slower.
  • On Monet, added breakpoint to start writing code to ID primers and secondaries that are highly repeated and killed by BLAST to abundant genes.
  • BLAST is extremely slow for this purpose. (30 min per attempted primer and secondary set). Investigating BLAT alternative.
  • probe names are non-unique.
    • should have recorded which pt number of the sequence the probe was from and this would not have been a problem
    • tried adding Tm. – no luck, still non-unique
    • adding instead random 12-number integer id per probe.
    • everything is unique. BLAST still refuses to build a database. darn it.

checking probe numbers:

  • variable length 4490 > 1, 5374 > .1, Tm 66-76
  • 35-55% GC content gives 4057 of 8750 with FPKM > 1

Multiplex chromatin

  • discussion with Bogdan on strategy 11:30 am 12:00 pm

Discussion with XZ

  • discussed setup distribution

Ph project

  • reran simulations on cluster size in Ph-ML to match expression range. Finished much faster this time.
  • zoomed in on sub-panels
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SDB 2015 abstract

Super-resolution Imaging of Chromatin Nanostructure
Links Epigenetic State and 3D Genome Packaging

Metazoan genomes are packaged at multiple scales, and regulation of this spatial organization may play an important role in the developmental and cell fate selection. Unfortunately, current methods provide little information about 3D organization of the genome at the length scale of genes (kilobases) and regulatory domains (hundreds of kilobases) in single cells. I will present a new super-resolution imaging approach to study the structural organization of the genome at the kilobase to megabase scale in individual cells at 20 nm resolution. These domains largely occupy diffraction limited volumes and thus their structures cannot be resolved with conventional imaging approaches. From thousands of images of dozens of epigenetic domains from across the Drosophila genome, we have discovered, within a single cell type, a substantial diversity of structural patterns: compact and diffuse domains, branched and linear domains, domains that are highly entangled with one-another and domains which are strictly segregated. These different structural features are closely correlated to certain differences in the epigenetic state of the chromatin. I will focus on the organization of Polycomb bound domains, which exhibit a surprising, entangled structure and length-dependent compaction. Computational models suggest this organization could contribute to the repressive nature of the domains. Preliminary comparisons between developing tissues suggests differential regulation of chromatin structure associated with developmental fate selection. This work suggests further super-resolution imaging studies of chromatin structure at this scale may greatly aid our understanding of the role of genome structural regulation in development.

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Monday 04/13/15

9:00 am – 6:20 pm, 8:00 pm- 10:30 pm

MERFISH

  • design new libraries
  • discussed design with team (9:30-10:30am)
  • working on library assembly (8:00pm-10:00pm)

New Libraries (140 gene).

  1. 20mer secondaries 30mer targeting regions 1-16 (shorter than before)
  2. 20mer secondaries 30mer targeting regions 17-32
  3. FAST 20mer secondaries 30mer targeting regions 1-16
  4. FAST 20mer secondaries 30mer targeting regions 1-16
  5. 20mer secondaries 6 on-bits 30mer targeting 1-16. (2 bits per target)
  6. 20mer secondaries 30mer targeting regions 1-16 (4 bits per target)

For later

  1. 20mer secondaries variable targeting

Chromatin tracing

  • Prep for chromatin tracing meeting 10:40 – 2:00pm
  • discussion with Bogdan on strategy 11am-12pm
  • see slides
  • tracing discussion with TW and XZ and team 2:00pm – 6:15 pm

Ph project

  • new draft of figures and text from NF

Abstract Submission

  • submitted abstract for SDB meeting, ~2 hours before deadline (I think). 10:00-10:30pm
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Saturday 04/11/15

Ph Project

  • working on simulations

Assume certain amount of open sites. All will be fill with no Ph.
Add PhM. The same fraction of PhM will be bound as the fraction of Ph bound, since the kinetics are the same.

start with 500 unbound and 1500 bound with no PhM, all sites occupied, eq constant 7:1 bound
as we add PhM, it fills and kills sites, only need 1 per site to kill it (if capped early). Sites tend to either fill to max or get capped as created.
Strong transition point at PhM = PhE/2.

Rendering

  • improved rendering: scatter is proportional to cluster size
  • should zoom in more — difficult because matlab makes chain look disconnected where it goes outside of frame.
  • should look up how to set zoom by command line (manual scroll does not work smoothly as it should).

Summary

  • See email to Nicole and Ajaz describing today’s results.
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Friday 04/10/15

10:00 am – 10:15 pm

group meeting 10am-1pm

MERFISH library design

  • would like a new human transcriptome database, build off of multiple cell lines.
  • Steven has ENCODE lines downloaded and cufflinks run. Just need to write matlab script to parse data

Ideas for increasing probe count:

  • allow flexible probe length

Ph simulations

  • wt can not join a cluster with equal Ph.
  • fixed bug in modification (empty sites still need to allow Ph to join)
  • started working on prettier model rendering for paper figures
    modelState(2)
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MERFISH published! (in Science Magazine)

http://www.sciencemag.org/content/early/2015/04/08/science.aaa6090.abstract

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