Kun:LabNotes/HiResChrPaint

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Project threads

Overview

Barcode design

  • Each barcode has N number of digits, each digit can have M colors. We start with N=7 and M=4.
  • To better resolve the barcodes, we require that two adjacent digits have different color. Therefore, the total number of possible barcodes is M*(M-1)^(N-1), which is 8748 when N=7 and M=4.
  • The size of a digit depends on how well we can resolve a feature optically. Based on existing publications, this could be anywhere between 10-400kb.
  • There could be a spacer between digits, but not necessary as long as two adjacent digits have different colors.
  • We do need a spacer between two adjacent barcodes in order to know where the first digit is. As a start, the spacer can be twice as large as a digit.
  • Here is an estimation of number of barcodes and probes needed for the entire human genome.
Digit Size # probes/digit Digits/barcode # barcodes Barcode size Total region covered # probes needed for human genome
50,000 400 5 324 350,000 113,400,000 648,000
6 972 400,000 388,800,000 2,332,800
7 2916 450,000 1,312,200,000 8,164,800
8 8748 500,000 4,374,000,000 18,560,000
100,000 400 5 324 700,000 226,800,000 648,000
6 972 800,000 777,600,000 2,332,800
7 2916 900,000 2,624,400,000 8,164,800
8 8748 1,000,000 8,748,000,000 9,280,000
200,000 400 5 324 1,400,000 453,600,000 648,000
6 972 1,600,000 1,555,200,000 2,332,800
7 2916 1,800,000 5,248,800,000 4,511,111
8 8748 2,000,000 17,496,000,000 4,640,000
  • As long as we can resolve individual barcodes, and two adjacent digits have different color, the mapping resolution equals to the size of a digit, not the size of a barcode.
  • We can also organize the distribution of the barcodes along the entire genome, such that there is a high-level pattern. For example, all barcodes in the same chromosome or chromosome bands can share the same first three digits, similar to how zip codes are designed.

Probe design

  • Criteria for a good probe:
    • Fixed probe length (easier for probe production), 70nt for now;
    • Relatively homogeneous Tm: calculate the Tm for a reasonably large pool of 70-mers, find the mean and STDEV, then set the range: 76-86C for now;
    • Unique across the genome: align each oligo to the genome with BLAT, the second hits (if exist) should have low homology;
    • No homopolymers: avoid oligos that have seven bases of the same in a row for now. The threshold of seven is somewhat arbitrary.
    • Low tendency to form hairpins or other secondary structures: calculate the folding energy using UNAFold, use a lower bound of -7 for now.

Probe synthesis and labeling

Hybridization and imaging

Image analysis

Daily Notes

2012 <calendar> name=Kun:LabNotes/HiResChrPaint format=%name/%year-%month-%day date=2012/02/01 view=oneyear </calendar>