Wednesday, July 21, 2021

One of these things doesn't belong: efforts to exclude problematic sequences in GRCh38

Since the release of GRCh38, the GRC has received a number of user reports alerting us to a potential false duplication involving chr 21p and 21q. Users noted that reads were aligning to both regions in GRCh38, but not GRCh37/hg19, resulting in a decreased mapping score and difficulties in variant calling throughout. Additionally, user analyses involving Multiplex Ligation-dependent Probe Amplification (MLPA), a technique for gene copy number detection, and exome studies indicated potential false duplications. The implicated regions contained several genes, including CBS (Gene ID: 875), U2AF1 (Gene ID: 7307) and KCNE1B (Gene ID: 3753). The GRC has investigated the matter and concurs that the GRCh38 assembly contains sequence on the short arm of chr 21 that should be excluded from analyses. Read on to learn more about this issue, as well as some recently detected non-human contamination in GRCh38, and ways you can find and avoid these sequences in your analyses.

The short arm of human chromosome 21, like that of the four other human acrocentric chromosomes, is where genes associated with rDNA synthesis are localized, and is characterized by highly repetitive heterochromatic sequence. The repetitive nature of these sequences, coupled with limitations in sequencing technology, have until recently made the representation of these regions in genome assemblies very difficult. 

As a consequence, the GRCh37 representation of the chromosome 21 p-arm contained only 11 clone sequences. Seven were clones from the HSA21-specific BAC library CHORI-507 that had previously been experimentally localized to 21p (PMID: 17895424). In an effort to add additional sequence to this repetitive region, 23 additional components were added to 21p for GRCh38, including 18 additional CHORI-507 clones, 4 RPCI-11 clones, and 1 ABC9 fosmid. Admixture mapping localized some of these clones to this region.

In response to the user reports, the GRC re-reviewed the sequences added to 21p in GRCh38. Haploid CHM13hTERT Illumina reads generated by The McDonnell Genome Institute were aligned to GRCh38 by NCBI, and evaluated for read mapping and coverage. This analysis supported the user reports, suggesting that 5 of the newly added CHORI-507 clones (FP565260.4, CU639417.17, FP236240.8, FP475955.4 and CU633980.13) were actually redundant with sequences on chr 21q, and thus represented false duplications in GRCh38.

The GRC has now removed these sequences from the files that it uses to generate the reference assembly. However, we cannot remove them from the GRCh38 assembly without triggering the next major release of the human assembly. In order to help users recognize these regions and avoid them in their analyses, we have produced a masking file to be used as a companion to GRCh38. This BED file is available from the GenBank FTP site: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.28_GRCh38.p13/GRCh38_major_release_seqs_for_alignment_pipelines/GCA_000001405.15_GRCh38_GRC_exclusions.bed. This file provides the assembly coordinates of the 5 clones incorrectly localized to chr 21p. The Genome in a Bottle Consortium recently posted a preprint demonstrating that using this masking file greatly improves variant calling accuracy in the affected genes (https://doi.org/10.1101/2021.06.07.444885).

In addition to these sequences, the file also includes 2 other assembly scaffolds that were found, after the release of GRCh38, to be contaminated with non-human sequence. These include a chr Un scaffold (KI270752.1/NT_187507.1), whose sole component (AF065393.1) is now known to represent sequence from Chinese hamster (PMID:30486838), likely derived from the human-hamster CHO cell line that was the clone source, and an alternate loci scaffold (KI270825.1/NT_187580.1) whose non-anchor component (AC225822.3) was shown to be chimeric. In AC225822.3, the first 25,375 bases are human sequence matching GRCh38 chr 10 reference component and alternate scaffold anchor sequence AL391421.27, while the rest match Acidithiobacillus thiooxidans sequences from multiple WGS projects (PMID:32398145). Although all sequences in the reference assembly are screened for foreign contamination, these two were not detected at the time of release (2014). Prompted by these findings, the GRC has more recently re-screened the assembly with updated contamination databases and has not detected additional issues. As these two scaffolds are not human sequence, very few reads are likely to map well to them, but users may still want to make note of them in their analyses.

In total, the contamination represents ~800 Kb, or 0.02% of the total sequence length. GRCh38 remains an extremely high quality reference assembly. Nonetheless, the GRC remains committed to addressing assembly errors and making sure it serves as the most reliable analysis substrate possible. Check out our website to see other genomic regions under review. We welcome your feedback and reports of newly discovered issues!  In the future, we plan to update the masking file with any new regions as identified and reviewed by the GRC.


 Fig. A

Fig. A:  Aligned CHM13hTERT Illumina reads viewed in Integrative Genomics Viewer (IGV). The panes labeled 'Original' were reads aligned prior to redundant sequence masking and the panes labeled 'Fixed' are reads aligned after redundant sequence masking.  

The top two panes show reads aligned to the valid U2AF1 locus in GRCh38 (NC_000021.9:43,091,000-43,110,000 of 21q) and the bottom two panes show reads aligned to the falsely duplicated (pseudo region) region of GRCh38 (NC_000021.9:6,480,000-6,500,000 of 21p). 

In IGV, sequence reads that align to 2 places in the Reference (whether it is correct or not), yield poor/ambiguous alignments, indicated by clear, unshaded reads. This is shown by both the 'Original hg38 U2AF1' and the 'Original hg38 pseudo region' pane. 

Following the masking of the known, duplicated region introduced in GRCh38, the aligned reads in the 'Fixed hg38 U2AF1 gene' pane are shaded grey, meaning they have good mapping scores to that region. And there are no reads mapping to the 'Fixed hg38 pseudo region' because the duplicated sequence is masked in the Fixed hg38 file.



Fig. B

Fig. B: Aligned reads to 21q region that has false duplication on 21p in GRCh38 before masking. Note the BAC clone boundary where alignment of falsely duplicated region in 21p starts. This duplication involves the CBS gene (Gene ID: 875).


Fig. C

Fig. C: Aligned reads to 21p region falsely duplicated (in Fig. B). You can see ambiguous read alignment and the falsely duplicated CBS gene annotated in the gene track.


Fig. D

Fig. D: Alignment of BAC FP236240.8 (redundant BAC added to 21p for GRCh38) to the corresponding valid region on 21q. Note the redundant BAC alignment to the region (bottom pane) and the valid read alignment depth (shown in middle pane). Since the region was falsely duplicated, read alignment in the region of redundant BAC alignment is poor.


































25 comments:

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    Replies
    1. CHIEF DR LUCKY HERBAL THERAPY
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      5: HEPATITIS B
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      7: HUMAN PAPILLOMAVIRUS (HPV)
      8: CANCER
      9: PAD
      10: PENIS ENLARGEMENT
      11: THYROID ETC..

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      Delete
  2. This comment has been removed by the author.

    ReplyDelete
  3. Glad to see this effort to fix the errors in GRCh38. I wanted to point out that 2 of the errors were identified and published by my collaborators and me. The Chinese hamster sequence was found while we were building the CHESS human gene database, and we described it in several talks and in Pertea et al 2018, https://pubmed.ncbi.nlm.nih.gov/30486838/. The Acidithiobacillus thiooxidans contamination was found by the Conterminator program, described in Steinegger and Salzberg 2020, https://pubmed.ncbi.nlm.nih.gov/32398145/ and highlighted in Figure 3 of that paper. I know that a blog isn't an academic paper, but it would be nice if you would acknowledge the sources.

    ReplyDelete
    Replies
    1. I see the PubMed references are there now! Thanks very much for including them.

      Delete
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    ReplyDelete
  5. When you are going to update to CHM13 v1.1, which is finally complete genome level?

    ReplyDelete
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  9. It’s indeed a miracle. I can’t believe I am finally free from herpes virus, am grateful to this powerful herbalist called Dr Kham.
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  10. Hi friends!
    I am Cox Manuel by name and l want to use this great opportunity to share with you some great and amazing testimony of how I was cured from HERPES Virus with the help of Dr Utu Herbal Medicine.
    Over 2 years now I have been suffering and living in pain with this deadly virus Herpes, fear grew in my mind everyday as I heard there is no cure for it, but to me I knew that medical drugs was not the only option in treating viruses and infection, my greatest problem was that I was stocked in pain as the symptoms has circulated over my body.
    Few Months ago I decided to get more information online concerning herpes,and while i was surfing the web, I came across some great testimonials from various people on how Dr Utu has been using his herbal medicine to cure different disease, at first I was shocked as i have never heard of such thing before, and also i doubted if it was true as it never came up on media, these people gave out the email address of Dr Utu and advised we contact him for any problem, though I was doubting him I decided to put a test on it, moreover it was the fist time for me to hear something like that, I copied the email address of Dr Utu and I sent him a message about my health status and I also requested for his medicine in order for me to get good health, he gave me a responded after some hours and he told me the procedure involved in order for me to get his medicine, he also said to me he was going to send me some herbal liquid medicine which was going to take for four weeks and after four weeks I will be totally cured.
    I went with Dr Utu and I followed exactly as i was told by this man, after 3 to 5 days of communication I eventually received a package from the mailing service and that was the medicine sent by Dr Utu.
    I started taking the medicine as I was directed by Dr Utu and after two days I also started seen some changes on my skin, and within a month l noticed that the rashes on my skin are cleared, l was surprised and l had a thought to myself again LIKE! IS THIS REALLY WORKING OUT? when l noticed this changes l contacted this Great Dr and l told him about it, he told me to go to the hospital and do some check up if the virus is still there, l actually went to the hospital on Friday to do some test on my blood and after 3 days the result came out that l was completely free from HERPES, still many people will not believe this, but it did really works.
    Dr Utu is a brave man and he is set to answer all human disease, today l am living in good health as just with a test l conquered my pain and sorrow, Please you can contact Dr Utu for any problem with the email below:
    drutuherbalcure@gmail.com

    ReplyDelete
  11. Hello everyone. Dr. Ehimare. Herbs is a good remedy for herpes Virus , I was a carrier of herpes virus and I saw a testimony on how Dr. Ehimare. cure Herpes Virus, I decided to contact him, I contacted him and he guided me. I asked him for solutions and he started the remedies for my health. After he finish he sent me the herbs which i took for 2 weeks before asking me to go for a check up and getting there i could not believe that i was confirm HSV 2 Negative after the test, Today i am so happy because i'm free from herpes disease with the help of Dr. Ehimare. Thank God now everything is fine, I'm cured by Dr. Ehimare herbal medicine, I'm very thankful to God for making it possible. you can reach him on his email, i strongly recommend him to any one out here looking for a cure.Contact Dr Ehimare via email: Drehimare3@gmail.com call or whatsApp him on+1 (267) 691-1087

    ReplyDelete
  12. I was once a victim of Herpes Virus. A good friend of mine who was cured from diabetes introduced me to a doctor named Herbal Doc. Kham from Africa. At first I refused to contact the doctor because of my past experience with most doctors on the internet. Later that day when surfing the internet I came across a testimony of a young lady from Alabama who was cured from Genital Herpes with Herbal Doc. Kham Herbal Medicine, so I decided to give the doctor a try, I contacted the doctor with the email on the testimony and after much discussion with him, he prepared the Herbal medicine which I paid for and he sent it to me which I received 5 days later through courier service and after receiving it he prescribed the usage for two weeks that I will be cured. Truly after using the Herbal medicine for two weeks I discovered that there was improvement on my health and the outbreaks I used to have were gone I decided to go for another test and my result was negative with no trace of the virus on my blood, you too can be cured with this herbal doctor Natural products, Email him on: dr.khamcaregiver@gmail.com but you can also Whatsapp him Via +2348159922297 Here is his Website for more Information https://dr-kham-herbal-home.webnode.com/ for easy conversations, He has cure for Diabetes, Cancer, Fibroid, Painful joints, Heart disease and Liver disease

    ReplyDelete
  13. GRCh38.p14 was planned for release in the second half of 2020. Any update?

    ReplyDelete
  14. Hi friends!
    I am Cox Manuel by name and l want to use this great opportunity to share with you some great and amazing testimony of how I was cured from HERPES Virus with the help of Dr Utu Herbal Medicine.
    Over 2 years now I have been suffering and living in pain with this deadly virus Herpes, fear grew in my mind everyday as I heard there is no cure for it, but to me I knew that medical drugs was not the only option in treating viruses and infection, my greatest problem was that I was stocked in pain as the symptoms has circulated over my body.
    Few Months ago I decided to get more information online concerning herpes,and while i was surfing the web, I came across some great testimonials from various people on how Dr Utu has been using his herbal medicine to cure different disease, at first I was shocked as i have never heard of such thing before, and also i doubted if it was true as it never came up on media, these people gave out the email address of Dr Utu and advised we contact him for any problem, though I was doubting him I decided to put a test on it, moreover it was the fist time for me to hear something like that, I copied the email address of Dr Utu and I sent him a message about my health status and I also requested for his medicine in order for me to get good health, he gave me a responded after some hours and he told me the procedure involved in order for me to get his medicine, he also said to me he was going to send me some herbal liquid medicine which was going to take for four weeks and after four weeks I will be totally cured.
    I went with Dr Utu and I followed exactly as i was told by this man, after 3 to 5 days of communication I eventually received a package from the mailing service and that was the medicine sent by Dr Utu.
    I started taking the medicine as I was directed by Dr Utu and after two days I also started seen some changes on my skin, and within a month l noticed that the rashes on my skin are cleared, l was surprised and l had a thought to myself again LIKE! IS THIS REALLY WORKING OUT? when l noticed this changes l contacted this Great Dr and l told him about it, he told me to go to the hospital and do some check up if the virus is still there, l actually went to the hospital on Friday to do some test on my blood and after 3 days the result came out that l was completely free from HERPES, still many people will not believe this, but it did really works.
    Dr Utu is a brave man and he is set to answer all human disease, today l am living in good health as just with a test l conquered my pain and sorrow, Please you can contact Dr Utu for any problem with the email below:
    drutuherbalcure@gmail.com...

    ReplyDelete
  15. I WANT TO USE THIS OPPORTUNITY  TO SAY A VERY BIG THANK YOU TO Dr Ogberaese, My name is Leonard Shirin i am so happy to share this testimony on how Dr.Ogberaese  healed me from genital herpes. i was detected with genital herpes around 10th may 2019, and ever since that day i have been looking for a way to end this virus from my body, but the more i try, the more i was told there was no solution. Few months ago i saw a testimony of some people on how Dr Ogberaese   cure them from HERPES VIRUS, i also heard about it on media when a young lady also gave her testimony saying Dr Ogberaese cured her oral herpes. I was left with no other hope than to contact Dr Ogberaese and give him a try. I contacted him  and told him what i was going through. He said he could heal me, that he is going to send me a herbal medicine and He sent the medicine as he promised. I took this herbal medicine for 1 week and it was obvious that his medicine was for real, because the herpes started clearing from my body. Later at  the 2 week, i went for medical check up and i was surprised when my doctor told me he found no trace of the virus in me again. At first when he told me, i was so shock and still did not believed until he gave me my test result. to make my story short i am now free from herpes, after many months of sorrow and my sickness is gone, i thank God for leading me to this man, and i want to also lead you to him if you have any illness like HIV, HPV, hepatitis B, asthma, cancer, Diabetes, COLD SCORE,TOOTH ACHE  FIBROID and AUTISM.  You can email him on drogberaese54@gmail.com or whats-app  him on +2348073818653.  visit him on his website:  https://dr-ogberaese-herbal-home.jimdosite.com

    ReplyDelete
  16. Are you battling with any health related issues or infection is not the end of your life, why not contact a herbal practitioner for cure Dr Oyagu has herbal remedies and permanent solution for all your health related problems. recommend him to you today only because i have also used his herbal medicine and it worked perfectly on my health problem, you can actually save yourself from giving your hard earned money to Scammers if only you can contact Dr Oyagu today for genuine and Permanent cure herbal products . Pls help share this post to save yourself and your love ones. Dr Oyagu is specialize in these areas, Permanent herbal supplements for anxiety and depression, HIV/AIDS, Diabetes, Epilepsy, Blood, Cancer, HPV, ALS, herpes etc. Email: oyaguherbalhome@gmail.com or visit his website https://oyaguspellcaster.wixsite.com/oyaguherbalhome Or Cell Whatsapp Number +2348101755322 thank you

    ReplyDelete
  17. HELLO everyone! Still don’t know the right words to express my Gratitude to the Great Dr Apala After been diagnosed with the Herpes for the passed 3years, i was given so many health prescription and advice with no improvement, I totally lost hope, until i found many testimonies of Great Dr Apala in an online research Like anybody would be, i advice any one that is living with herpes should Contact the only Great Dr Apala Email:dr.apalaherbalhome@gmail.com or WhatsApp ‪+2349150342282‬ today, because he has cure my Herpes totally He also have herbs medicine to cure the following diseases;
    CANCER
    EPILEPSY.
    HIV AIDS.
    DIABETICS

    ReplyDelete
  18. Good luck to anyone reading this true life story of mine, I Was Diagnosed With type 2 Herpes Virus Last year, And I Was Looking For Solution To Be Cured Luckily I Saw Testimonies On How Dr OYAGU Cure Herpes Virus I Decided To Contact Dr OYAGU I Contacted Him He Prepared A Herbal Medicine Portion And Sent It To Me, I Started The Herbal Medicine For My Health. He Gave Me Step By Step Instructions On How To Apply It, When I Applied It As Instructed, I Was Cured Of This Deadly Herpes Within 2 weeks, I Am Now Herpes Negative. My Brother And Sister I No That There Are So Many People That Have The Same Herpes Virus Please contact Dr OYAGU To Help You Too, And Help Me To Thank Dr OYAGU For Cure Me, I’m Cured By Dr. OYAGU Herbal Medicine, His Contact Email:oyaguherbalhome@gmail.com or visit his website https://oyaguspellcaster.wixsite.com/oyaguherbalhome Or Cell Whatsapp Number +2348101755322 thank you

    ReplyDelete
  19. CHIEF DR LUCKY HERBAL THERAPY
    Thank you very much for curing my Human Papillomavirus (HPV) totally. You are really a Godsent, I promise you that I will always recommend you. and to share your testimony to people. Contact him now thanks all.
    CHIEF DR LUCKY HERBAL THERAPY CAN AS WELL CURE THE FOLLOWING VIRUSES :-
    1: HIV/AIDS
    2: HSV
    3: LUPUS
    4: ALS
    5: HEPATITIS B
    6: DIABETES
    7: HUMAN PAPILLOMAVIRUS (HPV)
    8: CANCER
    9: PAD
    10: PENIS ENLARGEMENT
    11: THYROID ETC..

    WhatsApp him on +2348132777335 or email (chiefdrlucky@gmail.com).

    Website: https://chiefdrluckyherbaltherapy.wordpress.com/

    Http://facebook.com/chiefdrlucky

    ReplyDelete

  20. A great testimony that i must share to all HERPES patient in the world i never believed that their could be any complete cure for HERPES or any cure for HERPES, i saw people's testimony on blog sites of how Dr ONUWA prepare herbal cure and brought them back to life again. i use the HERPES DRUGS the doctor gave to me. Right now i can tell you that few months now i have not had any pain, delay in treatment leads to death. All thanks to Dr ONUWA herbal Solution you can contact him through WhatsApp number +2348115914591 or Email; dronuwa2@gmail.com





    https://dronuwa2.wixsite.com/my-site

    ReplyDelete
  21. DR OYAGU herbal medicine is a good remedy for herpes simplex virus cure. My name is MIGNON WRIGHT , am from USA,I am here to give my testimony about DR OYAGU who helped me in my life, i want to inform the public how i was cured from (HERPES SIMPLEX VIRUS) by DR OYAGU, i visited different hospital but they gave me list of drugs like Familiar, Zovirax, and Valtrex which is very expensive to treat the symptoms and never cured me. I was browsing through the Internet searching for remedy on HERPES and i saw comment of people talking about how DR OYAGU cured them, when i contacted him he gave me hope and send a Herbal medicine to me that i took and it seriously worked for me, am a free person now without any problem, my HERPES result came out negative, I pray to God to never let me and my family forget you in our life, am now cured permanently, you can also get your self cured my friends if you really need this doctor help, you can reach him on his website ;https://oyaguspellcaster.wixsite.com/oyaguherbalhome Email: oyaguherbalhome@gmail.com or via whatsapp +2348101755322

    ReplyDelete
  22. I am very happy today, I have been suffering from herpes since 2013, i have spent a lot on drugs and herbs which has all proved abortive, i have tried all means in life to cure my herpes , but there was no answer until i met  Dr.Oje Abacha   and i contacted him and he prepare medicine for me after I took his medication as he told me, i am now Herpes Negative, i am very very happy, thanks to  Dr.Oje Abacha for helping my life come back newly again without any form of crisis, i promise to tell your name and good deeds to the whole world. Kindly contact him for  cure regarding,   HIV, Herpes, Cancer and other deadly diseases. contact him on via oje.herbalsolutioncentre@gmail.com  or whatsApp him on +2348114715925  or His website  https://ojeherbalsolutioncentre.wordpress.com.

    ReplyDelete