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Tandem repeat expansion detection or genotyping from long-read alignments
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Straglr not finding TR's that are definitely present in targeted sequencing data #17

Closed AliSTaylor closed 8 months ago

AliSTaylor commented 1 year ago

Hello,

I'm testing Straglr out on some data from targeted sequencing sources. We can find the repeat regions with other repeat measuring programs and by examining the fasta/bam files. However, when straglr is run, no errors are shown but the output files are empty. We have tried by aligning to whole chromosome and gene-specific references, attempted on two separate sources and continue to get the same results.

Any help would be appreciated

readmanchiu commented 1 year ago

Thank you for trying Straglr and sorry to hear that nothing is output despite multiple efforts. I guess just I will list my questions/suggestions in point form so it may be clearer:

  1. Did you run it on ONT or PacBio sequences?
  2. Does your alignment BAM file contain the read sequences?
  3. Did you run it in genotyping mode (with --loci specfified with a bed file) or genome scan mode? Perhaps you can post the command you ran
  4. The empty result could either be resulted from: A) Straglr cannot extract the "target" region subsequence from the reads; B) the TRF result may not be deemed "proper" to be reported by Straglr
  5. For (A), it may happen with noisy ONT reads such that the boundary locations of the tentative repeat are not mapped in the alignment, hence Straglr fails to extract the sub-sequence. However, Straglr should output some warning messages indicating that it cannot extract sequences.
  6. For (B), the repeat sequence called by TRF does not span the majority of the extracted sub-sequence, suggesting the target locus contains more than the repeat, Straglr would chose not to report it.
  7. You could try running with the --debug mode on, which would keep the temporary files. I wonder if could see if there is a trf output (a .dat file). If there is, it should mean read sequences were extracted (not scenario A) and more likely scenario B.

There may be other scenarios but this should help starting the detective work.

AliSTaylor commented 1 year ago

Hello,

Thank you for your reply! I’ll answer each point by itself.

  1. It was run on PacBio CCS reads.
  2. The alignment BAM does contain the read sequences which have been measured using other programs (PacBio in-house, Tandem-Genotypes).
  3. Yes, it was run in genotyping mode, with loci specified. I also increased the range of the loci so that Straglr had a larger search area, after the first round of empty files. This is the command used: python straglr.py chr18.fasta —loci locus.bed 4-7. I’ve tried running the debug option. With one set of files, there was no output. With another set of files, there was output which I have attached.

The first attempt of this caused straglr to return an error of ValueError: start out of range (-495). I altered the bed file by 495nt to attempt to get some form of debug output which is attached. I’m unsure how to proceed here so any help would be appreciated.

On 14 Oct 2022, at 17:31, readmanchiu @.***> wrote:

Thank you for trying Straglr and sorry to hear that nothing is output despite multiple efforts. I guess just I will list my questions/suggestions in point form so it may be clearer:

Did you run it on ONT or PacBio sequences? Does your alignment BAM file contain the read sequences? Did you run it in genotyping mode (with --loci specfified with a bed file) or genome scan mode? Perhaps you can post the command you ran The empty result could either be resulted from: A) Straglr cannot extract the "target" region subsequence from the reads; B) the TRF result may not be deemed "proper" to be reported by Straglr For (A), it may happen with noisy ONT reads such that the boundary locations of the tentative repeat are not mapped in the alignment, hence Straglr fails to extract the sub-sequence. However, Straglr should output some warning messages indicating that it cannot extract sequences. For (B), the repeat sequence called by TRF does not span the majority of the extracted sub-sequence, suggesting the target locus contains more than the repeat, Straglr would chose not to report it. You could try running with the --debug mode on, which would keep the temporary files. I wonder if could see if there is a trf output (a .dat file). If there is, it should mean read sequences were extracted (not scenario A) and more likely scenario B. There may be other scenarios but this should help starting the detective work.

— Reply to this email directly, view it on GitHub https://github.com/bcgsc/straglr/issues/17#issuecomment-1279222266, or unsubscribe https://github.com/notifications/unsubscribe-auth/AGZW7LKH6BYIKWVR2GBA66TWDGDH7ANCNFSM6AAAAAARFIBJ4A. You are receiving this because you authored the thread.

problem getting seq1 m64231e_220404_135157/103351302/ccs ('htt_50repeats', 500, 981, 'CAG') None 1061 None problem getting seq1 m64231e_220404_135157/116524462/ccs ('htt_50repeats', 500, 981, 'CAG') None 1061 None problem getting seq1 m64231e_220404_135157/16910450/ccs ('htt_50repeats', 500, 981, 'CAG') None 1061 None trf input /tmp/tmpwyzwjhro too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/100205067/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/100795832/ccs 190 201 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/100926651/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/101058126/ccs 568 579 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/101386909/ccs 190 201 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/101843180/ccs 190 201 651 200 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189 200 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/118098783/ccs 578 585 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/118948598/ccs 189 200 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/119734601/ccs 579 584 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/120062938/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/120127568/ccs 578 583 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/120259150/ccs 578 585 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/120914187/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/121440687/ccs 189 200 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/122028168/ccs 580 585 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/122226805/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/122227034/ccs 187 198 637 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/122553842/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/122815402/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/122816664/ccs 579 584 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/12322858/ccs 188 199 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/123404534/ccs 581 586 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/124520607/ccs 203 216 659 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/1246927/ccs 588 595 652 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/124912884/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/125437029/ccs 580 585 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/126290010/ccs 188 199 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/127665692/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/127730869/ccs 583 590 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/127798027/ccs 589 594 652 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/128845283/ccs 188 199 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/128910494/ccs 188 199 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/129106834/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/129435733/ccs 523 534 643 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/129436217/ccs 580 585 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/130090410/ccs 192 203 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/130876945/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/13174748/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/132319168/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/132646541/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/133565188/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/134153624/ccs 189 200 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/134219099/ccs 189 200 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/134415466/ccs 189 200 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/134742399/ccs 582 587 643 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/135202413/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/135529250/ccs 188 198 637 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/135661101/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/137234983/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/137758424/ccs 576 587 647 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/138283456/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/138807707/ccs 192 203 645 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/139395440/ccs 430 435 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/139592089/ccs 306 319 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/139855531/ccs 189 200 645 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/141231737/ccs 191 207 657 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/141624714/ccs 466 473 645 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/142346178/ccs 188 199 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/142607875/ccs 578 583 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/142934970/ccs 190 201 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/142936201/ccs 188 199 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/143132148/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/14353712/ccs 189 200 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/143851898/ccs 189 200 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/144637988/ccs 578 585 643 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/146081161/ccs 520 531 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/146474061/ccs 520 531 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/147128472/ccs 188 199 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/147851138/ccs 190 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/147851359/ccs 579 586 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/148047859/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/148310363/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/148441598/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/148768693/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/150144351/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/150341175/ccs 196 206 648 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/151455731/ccs 188 198 638 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/151650842/ccs 191 202 643 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/151913900/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/152308031/ccs 432 437 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/152568259/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/152832614/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/154470742/ccs 577 582 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/154667229/ccs 189 200 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/154731199/ccs 193 204 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/155386752/ccs 188 199 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/155779981/ccs 556 561 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/156043677/ccs 522 533 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/156961328/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/157025459/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/157614736/ccs 577 582 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/15794281/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/158992531/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/16056997/ccs 189 200 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/160893270/ccs 580 585 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/160893542/ccs 191 202 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/161548868/ccs 188 199 638 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/161809991/ccs 554 564 645 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/162071814/ccs 469 476 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/162793860/ccs 573 584 647 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/162794242/ccs 581 586 643 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/162922842/ccs 188 200 630 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/162990135/ccs 93 104 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/163055037/ccs 190 201 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/163318274/ccs 188 198 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/163513184/ccs 191 202 645 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/163840084/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/16384422/ccs 188 199 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/163906326/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/163972393/ccs 602 607 670 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/165741488/ccs 188 199 638 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/16646305/ccs 192 203 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/166922287/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/167578367/ccs 104 115 650 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/168364112/ccs 520 531 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/168494943/ccs 188 199 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/168888622/ccs 581 592 653 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/169476259/ccs 190 201 644 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/170461921/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/171312152/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/171575009/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/172230879/ccs 189 200 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/173736305/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/174457582/ccs 555 561 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/174459112/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/175638730/ccs 554 560 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/17564291/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/175767716/ccs 587 592 651 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/175770460/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/17631756/ccs 580 585 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/176620752/ccs 189 200 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/176949867/ccs 189 200 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/17763044/ccs 578 585 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/177670610/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/17891713/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/17893244/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/18023508/ccs 579 584 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/18155459/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/18285163/ccs 576 583 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/18614464/ccs 189 200 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/19072585/ccs 587 592 649 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/19662815/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/20384286/ccs 188 200 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/21824203/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/22153490/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/23463003/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/23922065/ccs 190 201 646 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/25166882/ccs 594 601 662 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/25362892/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/25560496/ccs 191 202 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/2557497/ccs 307 321 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/25889166/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/27133590/ccs 520 531 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/27590696/ccs 190 201 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/27984838/ccs 580 585 645 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/27986422/ccs 186 197 639 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/29229399/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/30867515/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/31326411/ccs 190 201 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/32442063/ccs 189 200 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/32768728/ccs 189 200 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/3343629/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/33753897/ccs 189 200 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/34540382/ccs 190 201 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/34800280/ccs 581 586 643 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/35127917/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/35651603/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/36176263/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/36569151/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/36897834/ccs 189 200 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/37751457/ccs 521 532 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/38078044/ccs 189 200 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/38275330/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/38535478/ccs 188 199 642 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/38536991/ccs 188 199 641 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/38995411/ccs 188 199 640 200 too_far_from_read_end ('htt_50repeats', '500', '981') m64231e_220404_135157/39782346/ccs 188 199

readmanchiu commented 1 year ago

Thanks for posting the error messages. In the error message started with problem getting seq1 - the 4 items enclosed in the tuple after the read name should indicat the target locus: chromosome, start coordinate, end coordinate, and motif. Why is "htt_50repeats" the chromosome here? I saw you provided "chr18.fa" for the reference parameter. Shouldn't "chr18" be the chromosome name? as for the start and end coordinates of 500 and 981, are they genomic coordinates?

As for the errors started with too_far_from_read_end, I'm guessing by "altered the bed file by 495nt to attempt" you mean padding the target coordinates by 495. Please don't do that as the repeat reported by TRF has to be the majority of the sequence extracted from Straglr, i.e. if Straglr was told to extract the read sequence between A and B coordinates, and the TRF only detects a repeat between A+500, B-500, the result won't be deemed a positive one and ignored.

AliSTaylor commented 1 year ago

Hello,

Apologies for the misunderstanding. I’m trying straglr on 2 separate datasets. One dataset uses chromosome 18 as a reference. The other uses a version of the HTT gene with 50 repeats.

When assessing the samples aligned to the HTT gene, the coordinates are given relative to the reference sequence (i.e. 1-500) as the HTT repeat is close to the beginning of the gene.

As to the second part, yes, I tried originally not padding the coordinates but for some reason, Straglr will not analyse the data with the original genomic coordinates.

On 17 Oct 2022, at 17:47, readmanchiu @.***> wrote:

Thanks for posting the error messages. In the error message started with problem getting seq1 - the 4 items enclosed in the tuple after the read name should indicat the target locus: chromosome, start coordinate, end coordinate, and motif. Why is "htt_50repeats" the chromosome here? I saw you provided "chr18.fa" for the reference parameter. Shouldn't "chr18" be the chromosome name? as for the start and end coordinates of 500 and 981, are they genomic coordinates?

As for the errors started with too_far_from_read_end, I'm guessing by "altered the bed file by 495nt to attempt" you mean padding the target coordinates by 495. Please don't do that as the repeat reported by TRF has to be the majority of the sequence extracted from Straglr, i.e. if Straglr was told to extract the read sequence between A and B coordinates, and the TRF only detects a repeat between A+500, B-500, the result won't be deemed a positive one and ignored.

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readmanchiu commented 1 year ago

Let's focus on the one using genomic coordinates and genome alignments. When you ran with --debug on, did you get any error messages like problem getting seq1? Could it be the reference fasta you provided has difference chromsome names from the one you used for alignment? like one has a "chr" prefix and the other doesn't? If the answer is no for the above, it is feasible to share a subset of your data to debug?

AliSTaylor commented 1 year ago

Attachment available until 16 Nov 2022 Hello,

So no, they have the same chromosome name. I can’t attach that exact dataset. However, I have another dataset which we have published that I am experiencing the same issue with. It is a HTT dataset, which has been aligned to chromosome 4 which has genomic coordinates. It is PacBio data and results in empty files with no debugging information. I’ve attached it, along with the reference and the bed file. Click to Download https://www.icloud.com/attachment/?u=https%3A%2F%2Fcvws.icloud-content.com%2FB%2FAccDuXPDOJeZpDGbnrbRqs0xLRVjAeRvMz8VRDsTPe6Jv2MA1pfooDO8%2F%24%7Bf%7D%3Fo%3DAj1W3ETbGSU32TeJUaPkeLExSj9aN1VdMLrMvJEgH7Jh%26v%3D1%26x%3D3%26a%3DCAog4XVVtHgDdGDnChxNP3ncxS2ZjJHuUimJB-G0oU2SCIYSeBDe66W5vjAY3vugjcgwIgEAKgkC6AMA_z0wynVSBDEtFWNaBOigM7xqJsH9--qzOwJ3pGU3EZ8bAcclgxvWh5weH2TLZINJ6kTASs-9qwBxciY7ksY-N6EEw9ANNfJ5GVTVKp4-vQ8Gkh4w3XxGYfEumlc6xe5m2A%26e%3D1668622597%26fl%3D%26r%3DF765A839-0CD0-4147-8BAF-F9BD9EB9E984-1%26k%3D%24%7Buk%7D%26ckc%3Dcom.apple.largeattachment%26ckz%3D6111D02E-88BC-49DF-A7A4-52B3D8109BDB%26p%3D49%26s%3Dg_xcKjUsTpA8MU1N3ycid5hDP4w&uk=fifWVEQ70KVBlm5RIm_eHA&f=straglr_htt.tar.gz&sz=58712918straglr_htt.tar.gz 58.7 MB

On 17 Oct 2022, at 18:07, readmanchiu @. @.>> wrote:

Let's focus on the one using genomic coordinates and genome alignments. When you ran with --debug on, did you get any error messages like problem getting seq1? Could it be the reference fasta you provided has difference chromsome names from the one you used for alignment? like one has a "chr" prefix and the other doesn't? If the answer is no for the above, it is feasible to share a subset of your data to debug?

— Reply to this email directly, view it on GitHub https://github.com/bcgsc/straglr/issues/17#issuecomment-1281183090, or unsubscribe https://github.com/notifications/unsubscribe-auth/AGZW7LMJC4TWFGGTCK4KORLWDWBUDANCNFSM6AAAAAARFIBJ4A. You are receiving this because you authored the thread.

readmanchiu commented 1 year ago

Great, thanks, will take a look and get back to you

readmanchiu commented 1 year ago

OK, looked at the linked BAM, the alignments have a lot of clipped bases at either or both ends of the reads, Straglr won't interrogate these kinds of mappings. It will handle soft-clips, but only for alignment pairs where both ends of the reads are mapped, i.e. it will handle cases where a long repeat in the middle of a read leads to 2 soft-clipped alignments, but the ends of the read must be confidently mapped. The reads in this bam seems like kind of chimeric to me, where parts of the reads align to the same region multiple times. Of course I just cherry-picked some cases to look at, but that seems to be the reason why Straglr does not produce any report, because the alignments are all problematic.

AliSTaylor commented 1 year ago

I see. Thank you for the information! I’ll look into this with the other alignments.

On 17 Oct 2022, at 21:14, readmanchiu @.***> wrote:

OK, looked at the linked BAM, the alignments have a lot of clipped bases at either or both ends of the reads, Straglr won't interrogate these kinds of mappings. It will handle soft-clips, but only for alignment pairs where both ends of the reads are mapped, i.e. it will handle cases where a long repeat in the middle of a read leads to 2 soft-clipped alignments, but the ends of the read must be confidently mapped. The reads in this bam seems like kind of chimeric to me, where parts of the reads align to the same region multiple times. Of course I just cherry-picked some cases to look at, but that seems to be the reason why Straglr does not produce any report, because the alignments are all problematic.

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