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>