The .hic files are too small, inter.txt and inter_30.txt claim I only have inter-chromsomal reads

Ittai Eres

не прочитано,

17 окт. 2016 г., 17:41:0517.10.2016

– 3D Genomics

Hello,

I'm attempting to use juicer on a SLURM cluster to analyze some human Hi-C data.

After perusing the forums and doing some reading, I believe that perhaps there is something wrong with my directory structure and/or installation of juicer, but I can't seem to pinpoint it. Both my .hic files are extremely small (28M), and so are the inter.txt and inter_30.txt files (878 and 877 bytes, respectively). These latter two files say more or less the same thing: that I have no inter-chromosomal reads. Here's the text of inter.txt:

Experiment description:

Sequenced Read Pairs: 194,213,224

Normal Paired: 181,213,354 (93.31%)

Chimeric Paired: 8,740 (0.00%)

Chimeric Ambiguous: 14,517 (0.01%)

Unmapped: 12,976,613 (6.68%)

Ligation Motif Present: 2,103,772 (1.08%)

Alignable (Normal+Chimeric Paired): 181,222,094 (93.31%)

Unique Reads: 142,031,607 (73.13%)

PCR Duplicates: 39,165,471 (20.17%)

Optical Duplicates: 25,016 (0.01%)

Library Complexity Estimate: 356,280,767

Intra-fragment Reads: 87,177,128 (44.89% / 61.38%)

Below MAPQ Threshold: 27,710,260 (14.27% / 19.51%)

Hi-C Contacts: 27,144,219 (13.98% / 19.11%)

Ligation Motif Present: 100,298 (0.05% / 0.07%)

3' Bias (Long Range): 50% - 50%

Pair Type %(L-I-O-R): 0% - 0% - 0% - 0%

Inter-chromosomal: 27,144,219 (13.98% / 19.11%)

Intra-chromosomal: 0 (0.00% / 0.00%)

Short Range (<20Kb): 0 (0.00% / 0.00%)

Long Range (>20Kb): 0 (0.00% / 0.00%)

I've seen some mention of issues with the chrom.sizes file related to this problem--but I'm not entirely sure where this file is even supposed to be located (or if this would even be an issue when I am analyzing data with the "default" hg19 genome). I should note that I have only installed juicer on my cluster--I did not separately install juicebox; is this something that is required? Or is the juicebox_toools.7.5.jar sufficient when installed in setting up juicer?

I should note that I am also quite confident that I do have intra-chromosmal reads, as I have created heatmaps for these data in HOMER previously and the HiCUP processing report for these data find ~50 million unique di-tags, ~35 million of which are intra-chromosomal. So I think I have somehow merely used or installed juicer incorrectly...

Obviously with these extremely small/empty inter.txt and inter_30.txt files, my .hic files are mostly gibberish and the logs of the .hic jobs in /debug give me the error:

java.lang.RuntimeException: No reads in Hi-C contact matrices. This could be because the MAPQ filter is set too high (-q) or because all reads map to the same fragment.

at juicebox.tools.utils.original.Preprocessor$MatrixZoomDataPP.mergeAndWriteBlocks(Preprocessor.java:1397)

at juicebox.tools.utils.original.Preprocessor$MatrixZoomDataPP.access$000(Preprocessor.java:1168)

at juicebox.tools.utils.original.Preprocessor.writeMatrix(Preprocessor.java:582)

at juicebox.tools.utils.original.Preprocessor.writeBody(Preprocessor.java:313)

at juicebox.tools.utils.original.Preprocessor.preprocess(Preprocessor.java:223)

at juicebox.tools.clt.old.PreProcessing.run(PreProcessing.java:98)

at juicebox.tools.HiCTools.main(HiCTools.java:77)

Any help/guidance would be much appreciated!

Best,

Ittai Eres

Neva Durand

не прочитано,

18 окт. 2016 г., 02:23:0918.10.2016

– Ittai Eres, 3D Genomics

Hello Ittai,

The statistics indicate that all your intrachromosomal reads either mapped to the same fragment or were below the MAPQ threshold. You have 142,031,607 unique reads; 87,177,128 of these map to the same fragment (intra fragment) and 27,710,260 are MAPQ 0 (meaning they do not map uniquely).

In running Juicer, what did you use for the restriction site? Take a look at your merged_nodups file. The first 8 fields give strand1, chr1, pos1, frag1, strand2, chr2, pos2, frag2. If chr1 == chr2 and frag1 == frag2, the read ends map to the same fragment. We discard these reads as they are generally unligated DNA and do not represent Hi-C contacts.

Best

Neva

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Neva Cherniavsky Durand, Ph.D.

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www.aidenlab.org

Ittai Eres

не прочитано,

18 окт. 2016 г., 17:10:2618.10.2016

– 3D Genomics, redboy9...@gmail.com

Hi Neva,

Thanks for your prompt and helpful response. I took a look at my merged_nodups file, and all reads appear to be assigned to fragment 0. I am using MboI as the restriction enzyme...I did create the in silico restriction digest file myself with the provided python script, so perhaps something is wrong there, but looking at the hg19_MboI.txt file, it is 63 MB and contains lots of numbers, which is what I was expecting (it's just the genomic coordinates of MboI cut sites, right?).

Taking a closer look at the merged_nodups file, I don't understand why all the reads were assigned to fragment 0, since many of them come from drastically different genomic coordinates along the same chromosome. Below I've copied some of the beginning text from the merged_nodups file, and from the hg19_MboI.txt digest file:

merged_nodups:

0 chr1 10002 0 0 chr1 249240397 0 0 50M AACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA 0 50M GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 700819F:478:$

0 chr1 10009 0 0 chr1 249240403 0 0 50M ACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAAC 0 50M GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 700819F:478:$

0 chr1 10009 0 0 chr1 249240480 0 0 50M ACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAAC 0 50M TAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTA 700819F:478:$

0 chr1 10017 0 0 chr1 249240487 0 0 50M CCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCC 0 50M AGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG 700819F:478:$

0 chr1 10020 0 0 chr1 249240550 0 0 50M AACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA 0 50M TAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTA 700819F:478:$

0 chr1 10022 0 0 chr1 249240489 0 0 50M CCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACC 0 50M GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 700819F:478:$

0 chr1 10027 0 0 chr1 249240553 0 0 50M ACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAAC 0 50M GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG 700819F:478:$

0 chr1 10058 0 0 chr1 221535675 0 0 50M CCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACC 60 50M CTTTAAGGGTATACTGAAAACCCCTCTTACTTTTCACAGATACTGAAGGC 700819F:478$

0 chr1 10073 0 0 chr1 222853983 0 0 50M CAACCCTAACCCTAACCCTAACCCTAACCCTAACCCAACCCTAACCCTAA 60 50M AAAACACTTTATCAATGCAGCAATGGATCCAACAAAATCGAGAAATCGCA 700819F:478$

0 chr1 10092 0 0 chr1 249240563 0 0 17M1I32M AACCCTAACCCTAACCCAAACCCTAACCCTAACCCTAACCCTAACCCTAA 0 44M1D6M AGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAAGGTTAG 700$

0 chr1 10181 0 0 chr1 249240264 0 0 50M AACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA 0 43M7S GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTGGGGTGAGGG 700819F:47$

0 chr1 11123 0 0 chr1 379870 0 0 43M7S CTGAGACGGGTAGAACCTCAGTAATCCGAAAAGCCGGGATCGATCAAAAA 0 50M CTTTCACCTCCTTGGTTAAATGTACTCCTAAGTACAATCCTAAATGTGCT 700819F:478:H$

hg19_MboI.txt:

1 11160 12411 12461 12686 12829 13315 13420 13566 13698 13915 14377 15000 15172 15750 17954 18292 18376 18393 18512 18727 20029 20393 20477 20719 23312 23615 24082 25327 25925 26146 26390 26504 26595 26619 26849 27023 27945 27962 28115 29861 30603 30682 30830 31294 31518 31629 31647 32190 32262 32928 33130 34943 35425 35585 35642 36672 37146 37213 37657 38165 38346 38688 39073 39320 39398 39602 39617 39682 39857 39931 40014 40700 41859 42195 42394 42565 42650 42849 44117 44302 44723 45127 45408 46092 46589 46606 46881 47094 47641 47683 47809 48094 49189 49991 50991 51578 51643 51659 51885 52172 52658 53159 53282 54613 54962 54970 55004 55509 56626 56983 57141 57186 57443 57992 58382 58460 59234 59571 60078 60294 60303 60526 61826 62083 62099 62437 63095 63102 63191 63198 63323 63943 63952 64260 64840 65423 66082 66856 67305 67523 67847 68252 68903 69277 69408 69900 70008 70648 70689 72707 73546 74052 74401 74854 74903 75380 76385 76441 76544 76919 76951 77086 77639 77836 78149 78508 78796 78869 79320 79563 80179 80282 80859 80875 81034 81314 81716 81881 81945 82278 83199 83530 83619 84706 84741 84811 85627 85857 86942 87213 87352 88488 88561 88614 89194 89443 90687 90694 90900 90979 90995 91004 91154 91594 92410 92876 93002 93477 93948 93970 94278 95076 95522 95730 96022 96108 96275 97663 99336 99693 99716 100514 101445

Would this then suggest that there is perhaps something wrong with my juicer.sh script? I did have to change some things around to work with my specific cluster configurations and my directory structure, but I can't think of why else all the reads would map to the same fragment when the pairs are often from drastically different genomic locations.

Again, thank you for your prompt response and help on this! Really appreciative of the Aiden Lab making this resource public and helping users troubleshoot it :)

-Ittai

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Neva Durand

не прочитано,

18 окт. 2016 г., 17:15:2518.10.2016

– Ittai Eres, 3D Genomics

Hi Ittai,

The restriction site file should have the same name for chromosomes as in your aligned data. It looks like you aligned to a genome with "chr" in front of the chromosome name; so your restriction site file should also have that. Alternatively, you could get rid of all "chr" in merged_nodups (very easy with sed for example).

The reads you posted all have MAPQ 0 and would be skipped in any case (fields 9 and 12).

Let me know if you have any more questions.

Best

Neva

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Ittai Eres

не прочитано,

18 окт. 2016 г., 18:11:0418.10.2016

– 3D Genomics, redboy9...@gmail.com

Hi Neva,

That makes sense...not entirely sure why the restriction site file is missing the "chr" since I thought I made it myself previously with the hg19 file I downloaded that definitely has "chr" in front of the chromosome names, but oh well. I will make it again and try running through the pipeline once more to see if this resolves all the issues. I went ahead and checked that other reads in the merged_nodups file have MAPQ>0, just in case, so hopefully fixing this restriction digest file will solve everything. I'll get back to you once it's done.

Thank you so much!

-Ittai

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Neva Durand

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19 окт. 2016 г., 04:57:4019.10.2016

– Ittai Eres, 3D Genomics

Hi Ittai,

In the meantime, to see your data (but possibly with some intra fragment reads), you could run your merged_nodups file through the following:

awk '{$4=0; $8=1; print}' merged_nodups.txt > merged_nodups_fakefrag.txt

And then run the Juicebox command line tools “pre” command to make the .hic file (using this script):

juicebox pre merged_nodups_fakefrag.txt fakefrag.hic hg19

Best
Neva

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Ittai Eres

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21 окт. 2016 г., 18:52:0921.10.2016

– 3D Genomics, redboy9...@gmail.com

Hi Neva,

Thanks for your help so far...I ended up re-running the analysis after fixing my restriction digest file, and now I have a much more sensical merged_nodups.txt file that contains many different fragments.

However, now I'm running into an issue with creation of the .hic files--the debug error files for the hic and hic30 jobs both give me the following error:

Problem with creating fragment-delimited maps, NullPointerException.

This could be due to a null fragment map or to a mismatch in the chromosome name in the fragment map vis-a-vis the input file or chrom.sizes file.

Exiting.

As I said, I checked the merged_nodups file, and this time the fragments are different. That file specifies chromosome names with the "chr" abbreviation, as does my restriction digest file, and the Homo_sapiens_assembly19.fasta file that the juicer $genomePath points to (at least, so I believe--I didn't specify a genome in running juicer since I wanted it to default to hg19, but I looked through the script and double-checked that it points to where I think it does). Any other ideas? As I stated before, I'm uncertain about the path of the chrom.sizes file, so perhaps it has something to do with that? Or maybe the problem is in the inter.txt or inter_hists.m files? I've copied both of those below.

Thank you!

-Ittai

inter.txt:

Experiment description:

Sequenced Read Pairs: 194,213,224

Normal Paired: 181,213,354 (93.31%)

Chimeric Paired: 8,740 (0.00%)

Chimeric Ambiguous: 14,517 (0.01%)

Unmapped: 12,976,613 (6.68%)

Ligation Motif Present: 2,103,772 (1.08%)

Alignable (Normal+Chimeric Paired): 181,222,094 (93.31%)

Unique Reads: 142,082,949 (73.16%)

PCR Duplicates: 39,114,135 (20.14%)

Optical Duplicates: 25,010 (0.01%)

Library Complexity Estimate: 356,834,602

Intra-fragment Reads: 6,355,262 (3.27% / 4.47%)

Below MAPQ Threshold: 36,180,965 (18.63% / 25.46%)

Hi-C Contacts: 99,546,722 (51.26% / 70.06%)

Ligation Motif Present: 1,018,983 (0.52% / 0.72%)

3' Bias (Long Range): 67% - 33%

Pair Type %(L-I-O-R): 25% - 25% - 25% - 25%

Inter-chromosomal: 27,144,384 (13.98% / 19.10%)

Intra-chromosomal: 72,402,338 (37.28% / 50.96%)

Short Range (<20Kb): 35,987,875 (18.53% / 25.33%)

Long Range (>20Kb): 36,412,834 (18.75% / 25.63%)

inter_hists.m:

A = [

4287080 2571639 4256684 3461123 1746123 1410430 1261428 1195336 1131755 1074855 1035912 1025203 1037015 1029319 1013966 999251 939577 907799 870061 794740 735510 712763 677919 644046 643402 671455 688687 708603 761299 781237 827979 857764 877100 901968 922699 938396 934960 933861 941821 944814 946717 944696 949806 964136 964696 956410 958417 951479 947674 950750 945977 932625 951617 946910 932405 941280 933696 930928 914908 906378 911133 899634 906086 910443 910831 916445 913048 904305 894029 886039 877579 874714 867617 867668 866595 857186 851166 850505 835181 823178 815544 805349 800294 794275 791370 780373 774231 759538 746934 740034 731369 726900 723791 716771 713589 711631 707471 701864 696956 691251 686958 682386 677127 676036 672948 671650 668543 664920 659363 655666 649214 644291 642248 639511 635877 634078 633668 629808 628914 621351 619122 613464 608998 607146 604619 602068 598137 595867 594354 590836 586711 580743 577311 575716 571460 569786 564781 563848 560987 557850 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378 371 373 369 397 398 419 394 389 401 422 365 387 359 377 377 379 360 360 311 417 364 374 323 340 356 372 414 400 392 326 355 333 304 328 336 333 325 330 352 328 332 330 320 319 316 356 324 335 333 327 298 303 324 350 326 301 287 336 329 314 321 305 291 295 302 256 311 308 346 306 280 321 292 268 307 272 317 330 248 238 259 261 266 264 266 285 269 265 275 233 286 258 287 305 269 290 303 241 247 283 288 286 274 262 257 284 267 243 260 268 234 245 260 225 243 238 272 224 277 259 234 237 259 219 200 216 244 238 211 198 225 237 209 223 216 240 244 243 229 238 287 205 220 249 236 230 219 236 229 258 241 279 232 242 180 254 221 220 263 251 257 281 366 203 239 209 198 201 215 183 210 194 242 213 194 204 214 192 218 176 198 180 161 160 182 170 202 183 181 178 187 182 188 196 216 193 192 208 191 172 204 201 219 186 190 141 197 221 184 175 178 202 168 223 170 146 160 168 171 185 167 198 149 155 196 166 184 175 162 184 172 145 160 197 168 173 158 150 170 160 128 113 162 190 161 177 159 175 186 162 158 155 171 142 156 154 128 148 142 141 141 134 111 155 145 144 140 129 157 144 157 147 153 144 152 155 133 123 146 128 127 138 169 135 137 149 149 115 135 125 121 130 120 151 127 155 150 121 126 188 157 125 136 152 139 123 120 112 126 142 102 113 123 117 113 111 132 107 112 114 126 124 110 110 123 118 113 94 108 141 122 101 103 98 108 109 118 117 106 109 105 97 95 108 99 92 85 95 106 109 102 112 109 104 111 124 98 124 121 101 122 89 135 107 103 84 71 99 96 95 90 98 79 85 98 77 94 77 70 110 112 131 100 125 128 148 94 91 84 109 144 113 126 102 111 92 85 75 102 87 91 96 126 105 105 102 87 83 103 101 97 111 96 98 65 82 87 81 63 56 99 74 65 72 78 55 94 67 77 76 87 78 97 68 75 70 79 97 100 81 92 120 106 89 85 98 122 87 123 111 82 100 78 79 69 83 72 81 79 60 109 74 85 74 62 77 98 75 79 81 74 76 66 72 82 76 75 69 65 73 70 55 69 60 70 40 72 75 78 63 72 64 87 56 70 71 66 56 65 57 48 60 75 69 64 67 69 76 60 76 58 62 65 53 69 69 67 57 69 60 79 81 59 64 86 79 71 67 78 54 59 73 45 48 52 67 52 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];x = [

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];

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Neva Durand

не прочитано,

22 окт. 2016 г., 04:30:2522.10.2016

– Ittai Eres, 3D Genomics

Hi Ittai,

In this case, you probably need to run with your own chrom.sizes file. We’ve made a change so that the "chr" versus not won’t result in a problem, but I don’t think we’ve pushed it out yet.

You can easily make your own chrom.sizes from your fragment file via

awk 'BEGIN{OFS="\t"}{print $1,$NF}' restriction_site_file > hg19.chrom.sizes

Then call Juicebox tools with the hg19.chrom.sizes file for the genomeID.

juicebox pre -f restriction_site_file -s inter.txt -g inter_hists.m merged_nodups.txt inter.hic hg19.chrom.sizes

Best
Neva

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