Kun:LabNotes/HiResChrPaint: Difference between revisions

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**Different probe density: 50/digit; 200/digit; 400/digit
**Different probe density: 50/digit; 200/digit; 400/digit
**Different barcode size/spacing: 20kb/10kb; 40kb/20kb; 100kb/50kb; 400kb/200kb
**Different barcode size/spacing: 20kb/10kb; 40kb/20kb; 100kb/50kb; 400kb/200kb
**The regions were hand-picked. [[Media:chr19_various_spacing_probe_density_barcode_info.txt]]


===Probe synthesis and labeling===
===Probe synthesis and labeling===

Revision as of 02:33, 5 March 2012

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.
  • Update [2/22/2012]
    • It turns out that one limiting factor is that few good probes can be found for some genomic regions. When the probe length is reduced to 60nt, more good probes were identified. So we will switch to 60-mer probes, and the Tm is reduced to 73-83C.
    • My first attempt in getting enough probes for a list of genomic regions wasn't quite successful, because there were just not enough probes for many regions. Therefore, I decided to change the probe design strategy. I wrote a script findAllProbesInChr.pl to identify all good probes that can densely cover every chromosome first. From the list of good probes, I'll then do partitioning and trimming to assign probes to digits and barcodes.

Step 1: Find all good probes that densely cover the genome.

  • Perl Script: findAllProbesInChr.pl. This is the most time consuming step. It can take days on large chromosomes. We do it one chromosome at a time. The probe sequences can be used for a variety of barcoding schemes, so we only need to run the program for all chromosome once.
 ./findAllProbesInChr.pl /home/kunzhang/HsGenome/hg19.masked/chr15.fa.masked > chr15_probe_search.log &

Step 2: Group the probes into digits based on the digit size, spacing, and probe density.

  • Perl Script: probes2digits.pl. In the case that there are more than one possible way to group the oligos, all possibilities are evaluated exhaustively and the best one is chosen.
 ./probes2digits.pl 200000 100000 100 <  chr15_probe_seq.txt > chr15_probe_grouped_by_digits_200k_100k_100.txt &

Step 3: Group the digits into barcodes.

  • Perl Script: digits2barcodes.pl. Similar to the previous step, all possible combination of barcodes are evaluated to find the best one.
 ./digits2barcodes.pl 7 400000 < chr15_probe_grouped_by_digits_200k_100k_100.txt > chr15_probe_grouped_by_7_digits_barcodes_sp400k.txt

Step 4: Create a set of barcodes and allocate them to the genome.

 ./allocateBarcodeSpace.pl 7 100000 200000 > HsGenomeBarcodeAllocation.txt

Step 5: Assign the allocated barcodes to digits and probes.

./assignBarcodeColor.pl HsGenomeBarcodeAllocation.txt chr15_probe_grouped_by_7_digits_barcodes_sp400k.txt chr15_7_digits_barcodes_sp400k

Step 6: Add adaptors to the probes, group them based on fluorophores.

./probes2oligoPools.pl < chr15_7_digits_barcodes_sp400k_probe_seq_color.txt > chr15_oligoPools.txt

A master script to link everything together.

Genomic regions to target in the first probe set

  • Hox gene cluster: chr7:27.12M-27.30M. This is very small, will select barcodes that cover the region 25M-29M, 2,800 probes in total.
Barcode Barcode position Digit offset Digit ID Digit position Digit color
3421234 chr7:25092383-27094188 1 85 chr7:25092383-25292367 3
3421234 chr7:25092383-27094188 2 86 chr7:25392752-25592739 4
3421234 chr7:25092383-27094188 3 87 chr7:25692908-25892831 2
3421234 chr7:25092383-27094188 4 88 chr7:25992844-26192792 1
3421234 chr7:25092383-27094188 5 89 chr7:26294006-26493909 2
3421234 chr7:25092383-27094188 6 90 chr7:26594282-26794247 3
3421234 chr7:25092383-27094188 7 91 chr7:26894433-27094188 4
3421241 chr7:27494794-29492367 1 93 chr7:27494794-27693444 3
3421241 chr7:27494794-29492367 2 94 chr7:27793569-27993512 4
3421241 chr7:27494794-29492367 3 95 chr7:28093548-28292337 2
3421241 chr7:27494794-29492367 4 96 chr7:28392552-28592526 1
3421241 chr7:27494794-29492367 5 97 chr7:28692735-28892703 2
3421241 chr7:27494794-29492367 6 98 chr7:28992787-29192364 4
3421241 chr7:27494794-29492367 7 99 chr7:29292653-29492367 1
  • Chr15 regions based on HiC data: will use whole-genome barcodes in the entire chr15 (39,160 probes);
    • As a backup plan, I also manually picked digits in these regions for a second probe set that has a different amplification primer pair (V4). 11,759 probes in total
  Domain  ChrStart      ChrEnd      Size       Digit IDs(color)      
    A    42,252,708   42,772,708   520,000     66(1),67(1),68(1)
    A    44,092,708   44,852,708   760,000     72(2),73(2),74(2)
    B    52,532,708   53,052,708   520,000     100(3),101(3),102(3)
    B    58,492,708   59,212,708   720,000     119(4),120(4),121(4)
    A    67,172,946   68,092,946   920,000     148(1),149(2),150(1),151(2)
    A    76,252,945   77,212,945   960,000     179(2),180(1),181(2)
    B    83,202,945   84,848,996   1,646,051   200(3),201(4),202(1),203(3),204(4),205(1)
    B    93,598,996   94,918,996   1,320,000   234(4),235(2),236(3),237(2),238(4)
    ./extractProbesInDigits.pl chr15_HiC_region_digit_info.txt chr15_probe_grouped_by_digits_200k_100k_100.txt > chr15_HiC_region_oligoPools.txt
  • Chr3 region that might be inverted in African: 45M-60M, 15,226 probes in total. To fill out the space of 80,000 oligos, I decided to expand the region to 20M-66M, and include 26,281 probes
  • Combined all the oligos and split them based on the color, I ended up getting four pools with different sizes. This is because with the way I assign the barcodes different chromosomes (or regions) have enrichment on different colors, which leads to the unbalanced distribution when design probes on a small number of chromosomal regions.
 19518 Feb2012_80k_oligoPools.color_1.txt
 28967 Feb2012_80k_oligoPools.color_2.txt
 15400 Feb2012_80k_oligoPools.color_3.txt
 16114 Feb2012_80k_oligoPools.color_4.txt

Since each pool contains 20k sequences, I will have to exclude 8967 oligos in color 2. These oligos are from the end of chromosome 15.

  • Because the pool sizes for the other three colors are less than 20k, I decided to use the space to test additional ideas. These oligos will also be amplified with the V4 primer pairs, so that we can separate them from the whole genome set.

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>