Kun:LabNotes/HiResChrPaint: Difference between revisions

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*[[Media:chrPaintingProbeDesignMaster.txt|chrPaintingProbeDesignMaster.pl]]. To complete all steps within a reasonable time frame, Step 1 should be run on different chromosomes in parallel.
*[[Media:chrPaintingProbeDesignMaster.txt|chrPaintingProbeDesignMaster.pl]]. To complete all steps within a reasonable time frame, Step 1 should be run on different chromosomes in parallel.
====Genomic regions to target in the first probe set====
====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.
*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.
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Barcode'''
| align="center" style="background:#f0f0f0;"|'''Barcode'''
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*Chr15 regions based on HiC data: will use whole-genome barcodes that cover these regions;  
*Chr15 regions based on HiC data: will use whole-genome barcodes that cover these regions;  
*As a backup plan, I also manually picked digits in these regions for a second probe set that has a different amplification primer pair (V6).
**As a backup plan, I also manually picked digits in these regions for a second probe set that has a different amplification primer pair (V6). 11,759 probes in total
   Domain  ChrStart      ChrEnd      Size      Digit IDs(color)       
   Domain  ChrStart      ChrEnd      Size      Digit IDs(color)       
     A    42,252,708  42,772,708  520,000    66(1),67(1),68(1)
     A    42,252,708  42,772,708  520,000    66(1),67(1),68(1)
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     ./[[Media:extractProbesInDigits.txt|extractProbesInDigits.pl]] [[Media:chr15_HiC_region_digit_info.txt|chr15_HiC_region_digit_info.txt]] chr15_probe_grouped_by_digits_200k_100k_100.txt > chr15_HiC_region_oligoPools.txt
     ./[[Media:extractProbesInDigits.txt|extractProbesInDigits.pl]] [[Media:chr15_HiC_region_digit_info.txt|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.
*Chr3 region that might be inverted in African: 45M-60M, 15,226 probes in total.
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Barcode'''
| align="center" style="background:#f0f0f0;"|'''Barcode'''

Revision as of 23:57, 28 February 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 that cover these regions;
    • As a backup plan, I also manually picked digits in these regions for a second probe set that has a different amplification primer pair (V6). 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.
Barcode Barcode position Digit offset Digit ID Digit position Digit color
2432342 chr3:39094053-41110420 1 134 chr3:39094053-39294024 2
2432342 chr3:39094053-41110420 2 135 chr3:39395817-39592294 4
2432342 chr3:39094053-41110420 3 136 chr3:39693516-39893504 3
2432342 chr3:39094053-41110420 4 137 chr3:39994518-40194104 2
2432342 chr3:39094053-41110420 5 138 chr3:40294406-40494209 3
2432342 chr3:39094053-41110420 6 139 chr3:40610902-40810599 4
2432342 chr3:39094053-41110420 7 140 chr3:40910637-41110420 2
2432343 chr3:41517138-43521158 1 142 chr3:41517138-41716124 2
2432343 chr3:41517138-43521158 2 143 chr3:41822590-42020548 4
2432343 chr3:41517138-43521158 3 144 chr3:42120706-42320291 3
2432343 chr3:41517138-43521158 4 145 chr3:42421015-42620373 2
2432343 chr3:41517138-43521158 5 146 chr3:42721270-42919068 3
2432343 chr3:41517138-43521158 6 147 chr3:43019413-43219400 4
2432343 chr3:41517138-43521158 7 148 chr3:43321164-43521158 3
2432412 chr3:44220966-46068915 1 151 chr3:44220966-44416532 2
2432412 chr3:44220966-46068915 2 152 chr3:44517551-44717545 4
2432412 chr3:44220966-46068915 3 153 chr3:44817589-45017143 3
2432412 chr3:44220966-46068915 4 154 chr3:45117716-45316736 2
2432412 chr3:44220966-46068915 5 155 chr3:45425424-45625251 4
2432412 chr3:44220966-46068915 6 156 chr3:45725403-45925360 1
2432412 chr3:44220966-46068915 7 157 chr3:46025488-46068915 2
2432413 chr3:46477241-48267440 1 159 chr3:46477241-46677230 2
2432413 chr3:46477241-48267440 2 160 chr3:46777580-46804284 4
2432413 chr3:46477241-48267440 3 161 chr3:46866976-47066959 3
2432413 chr3:46477241-48267440 4 162 chr3:47167078-47367048 2
2432413 chr3:46477241-48267440 5 163 chr3:47467165-47665382 4
2432413 chr3:46477241-48267440 6 164 chr3:47767766-47966811 1
2432413 chr3:46477241-48267440 7 165 chr3:48067443-48267440 3
2432414 chr3:48668056-50670036 1 167 chr3:48668056-48864660 2
2432414 chr3:48668056-50670036 2 168 chr3:48964739-49164729 4
2432414 chr3:48668056-50670036 3 169 chr3:49271517-49470947 3
2432414 chr3:48668056-50670036 4 170 chr3:49571036-49770485 2
2432414 chr3:48668056-50670036 5 171 chr3:49870761-50069947 4
2432414 chr3:48668056-50670036 6 172 chr3:50170201-50369876 1
2432414 chr3:48668056-50670036 7 173 chr3:50470167-50670036 4
2432421 chr3:51075040-52745795 1 175 chr3:51075040-51274994 2
2432421 chr3:51075040-52745795 2 176 chr3:51347235-51545272 4
2432421 chr3:51075040-52745795 3 177 chr3:51645455-51772685 3
2432421 chr3:51075040-52745795 4 178 chr3:51805544-52005525 2
2432421 chr3:51075040-52745795 5 179 chr3:52105609-52305538 4
2432421 chr3:51075040-52745795 6 180 chr3:52405556-52604578 2
2432421 chr3:51075040-52745795 7 181 chr3:52705061-52745795 1
2432423 chr3:53366933-55376522 1 184 chr3:53366933-53566927 2
2432423 chr3:53366933-55376522 2 185 chr3:53667598-53867572 4
2432423 chr3:53366933-55376522 3 186 chr3:53970456-54170444 3
2432423 chr3:53366933-55376522 4 187 chr3:54270503-54469400 2
2432423 chr3:53366933-55376522 5 188 chr3:54569708-54769626 4
2432423 chr3:53366933-55376522 6 189 chr3:54869726-55069679 2
2432423 chr3:53366933-55376522 7 190 chr3:55176638-55376522 3
2432424 chr3:55776781-57776122 1 192 chr3:55776781-55976734 2
2432424 chr3:55776781-57776122 2 193 chr3:56076800-56275739 4
2432424 chr3:55776781-57776122 3 194 chr3:56375906-56575183 3
2432424 chr3:55776781-57776122 4 195 chr3:56675228-56875147 2
2432424 chr3:55776781-57776122 5 196 chr3:56975155-57174807 4
2432424 chr3:55776781-57776122 6 197 chr3:57275178-57475080 2
2432424 chr3:55776781-57776122 7 198 chr3:57576267-57776122 4
2432431 chr3:58473933-60473336 1 201 chr3:58473933-58673830 2
2432431 chr3:58473933-60473336 2 202 chr3:58773975-58972828 4
2432431 chr3:58473933-60473336 3 203 chr3:59073164-59273004 3
2432431 chr3:58473933-60473336 4 204 chr3:59373123-59572158 2
2432431 chr3:58473933-60473336 5 205 chr3:59672171-59872034 4
2432431 chr3:58473933-60473336 6 206 chr3:59972949-60172898 3
2432431 chr3:58473933-60473336 7 207 chr3:60273368-60473336 1
2432432 chr3:60873367-62875355 1 209 chr3:60873367-61073254 2
2432432 chr3:60873367-62875355 2 210 chr3:61177251-61375747 4
2432432 chr3:60873367-62875355 3 211 chr3:61476255-61676247 3
2432432 chr3:60873367-62875355 4 212 chr3:61776339-61976272 2
2432432 chr3:60873367-62875355 5 213 chr3:62076754-62275054 4
2432432 chr3:60873367-62875355 6 214 chr3:62375328-62575130 3
2432432 chr3:60873367-62875355 7 215 chr3:62675359-62875355 2

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>