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Thread: Problems with Illumina whole exome testing results

  1. #11
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    I got the Balaton ref from a more recent review of the X by the same author. May not answer your questions but I found it very interesting.

    The eXceptional nature of the X chromosome
    Bradley P Balaton Thomas Dixon-McDougall Samantha B Peeters Carolyn J Brown Author Notes
    Human Molecular Genetics, Volume 27, Issue R2, 01 August 2018, Pages R242–R249, https://doi.org/10.1093/hmg/ddy148
    Published: 26 April 2018

    "Abstract
    The X chromosome is unique in the genome. In this review we discuss recent advances in our understanding of the genetics and epigenetics of the X chromosome. The X chromosome shares limited conservation with its ancestral homologue the Y chromosome and the resulting difference in X-chromosome dosage between males and females is largely compensated for by X-chromosome inactivation. The process of inactivation is initiated by the long non-coding RNA X-inactive specific transcript (XIST) and achieved through interaction with multiple synergistic silencing pathways. Identification of Xist-interacting proteins has given insight into these processes yet the cascade of events from initiation to maintenance have still to be resolved. In particular, the initiation of inactivation in humans has been challenging to study as: it occurs very early in development; most human embryonic stem cell lines already have an inactive X; and the process seems to differ from mouse. Another difference between human and mouse X inactivation is the larger number of human genes that escape silencing. In humans over 20% of X-linked genes continue to be expressed from the otherwise inactive X chromosome. We are only beginning to understand how such escape occurs but there is growing recognition that escapees contribute to sexually dimorphic traits. The unique biology and epigenetics of the X chromosome have often led to its exclusion from disease studies, yet the X constitutes 5% of the genome and is an important contributor to disease, often in a sex-specific manner."

    https://academic.oup.com/hmg/article/27/R2/R242/4986911
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  3. #12
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    Quote Originally Posted by Ysearcher View Post
    yet the whole exome data indicates a diploid X chromosome, and the proband has a fatal neurodegenerative disorder. What am I missing here?? Either there IS a diploid X chromosome, or the data is incorrect. Which is it? I suspect lab error.
    Sorry our posts crossed. There are two issues here, how calls are reported (the 9/9 format is quite common in X vcfs unless you specifically suppress it during variant calling) and the proportion of 0/1 to 1/1s inside the X. The first issue can be ignored. Can you tally up 0/1s and 1/1s in the X chr from the vcfs?
    YSEQ:#37; YFull: YF01405 (Y Elite 2013)
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  4. #13
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    @MacUalraig, thanks much for your response.
    So your primary question is about the validity of heterozygous calls on a male X?
    Yes, but not just about heterozygous calls on the X, but the QUANTITY of heterozygous calls on the X. I have corresponded off and on with Dr. Turner from time to time, dating back to about ten years ago when she was the moderator of the Genealogy-DNA Rootsweb forum. I discussed these whole exomes with her for a few days when I first got the data more than five years ago, but about a completely different matter relating to one specific variant. I the time, I had no idea how to read the VCF format and made a lot of errors trying to interpret it. Thanks for the links, and I'll take a look ASAP, but I have already used up my allotted time today.

    With a little more searching over the weekend, I found this discussion - https://www.researchgate.net/post/Is...n_X_chromosome I liked the last response of that discussion, quoted -
    I agree with suggestions based on heterozygosity. I have done the following:
    Chart the fraction of heterzygous (0/1) calls per chromosome. In males, there will be a marked difference in this fraction. See the linked image from an individual genome.
    This is more straightforward and less variable of a method than copy number approaches or read depth because these can be complicated by reads piling up in low complexity regions or by differences in sequencing depth.
    http://3.bp.blogspot.com/-XpfNP7lHuA...22XY.karyo.png
    So I followed the suggestion, using whole exome data for proband and mother, and 23andMe data for proband, proband's sister and proband's paternal grandmother. Unfortunately, no 23andMe data for proband's mother, but never the less the results are interesting. Copied and pasted from my notes -

    Proband - (whole exome) X chromosome, after removal of pseudoautosomal regions
    total variants listed - 823
    heterozygous variants (0/1) - 197
    heterozygous variants (1/2) - 8
    homozygous variants (1/1) - 618
    fraction of heterozygous variants (205/823) - 0.2490887 (24.91%)
    fraction of homozygous variants (618/823) - 0.7509113 (75.09%)

    Proband's mother - (whole exome) X chromosome, after removal of pseudoautosomal regions
    total variants listed - 960
    heterozygous variants (0/1) - 599
    heterozygous variants (1/2) - 4
    homozygous variants (1/1) - 357
    fraction of heterozygous variants (603/960) = 0.628125 (62.81%)
    fraction of homozygous variants (357/960) = 0.371875 (37.19%)

    (Proband's sister) - 23andMe data (v2) - X chromosome, after removal of pseudoautosomal regions
    total variants listed - 13065
    heterozygous variants - 8889
    homozygous variants - 4176
    fraction of heterozygous variants - (8889/13065) = 0.68036739 (68.037%)
    fraction of homozygous variants - (4176/13065) = 0.31963261 (31.312%)

    Proband's paternal grandmother - 23andMe data (v2) - X chromosome, after removal of pseudoautosomal regions
    total variants listed - 13065
    heterozygous variants - 9000
    homozygous variants - 4065
    fraction of heterozygous variants - (9000/13065) = 0.68886338 (68.88%)
    fraction of homozygous variants - (4065/13065) = 0.31113662 (31.12%)

    As you can see, I subtracted the PARs before doing the calculations. The obvious difference between the whole exome data versus the 23andMe data is that the 23andMe data includes reference value homozygous calls, and the whole exomes do not. Also, it is an enriched whole exome, so it is very "skinny" - includes only a small fraction of known variants. Even so, the calculations are interesting. The image that I found from the researchgate forum certainly doesn't look anything like the proband's filtered X results. Proband's filtered heterozygous calls are 24.91%, proband's mother - 62.81%, and from 23andMe, proband's sister 68.03% (about the same as proband's paternal grandmother, 68.88%). I don't really understand why the whole exome technology shows so many heterozygous calls for (all) males on the X. Definitely a glitch in the technology, but what I'm trying to figure out is if 24.9% is higher than average, or pretty near normal. I think the only way I'm going to figure that out is to repeat this experiment with whole exome data from Open Human males. Probably another 12-15 hours of work.

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    Thanks for the reference. I have been doing quite a lot of research related to X inactivation. It's really difficult to understand all the various processes involved, and far from well defined by anyone. XACT is another important long non coding RNA involved in inactivation - https://www.genecards.org/cgi-bin/ca...&keywords=XACT

    Entrez Gene Summary for XACT Gene
    This gene produces a spliced long non-coding RNA that is thought to play a role in the control of X-chromosome inactivation (XCI). This transcript has been shown to specifically coat the active X chromosome in human pluripotent cells. [provided by RefSeq, Mar 2015]

    GeneCards Summary for XACT Gene
    XACT (X Active Specific Transcript) is an RNA Gene, and is affiliated with the non-coding RNA class.

    https://www.ncbi.nlm.nih.gov/pubmed/27989768
    Our findings therefore suggest a mechanism involving antagonistic activity of XIST and XACT in controlling X chromosome activity in early human embryos, and they highlight the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms.
    ,,,this raises the hypothesis that XACT function might be linked to the lack of tight control of XIST expression in early human development. In this scenario, XACT might have evolved to prevent X chromosome silencing and functional nullisomy and to permit an alternative strategy of dosage compensation at these critical developmental stages.
    https://www.omim.org/entry/300901
    XACT is a long intergenic noncoding RNA (lincRNA) that is expressed from and coats the active X chromosome (Xa) specifically in human pluripotent cells (Vallot et al., 2013).
    That last quote is what interests me about XACT - it is expressed from the active X. I have been trying to figure out for some time how to deduce from genomic data for related females, which haplotype is being expressed.

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    Posts crossing again.
    and the proportion of 0/1 to 1/1s inside the X. The first issue can be ignored. Can you tally up 0/1s and 1/1s in the X chr from the vcfs?
    I hope you got my first post today, which answers that question. Unfortunately, I am just out of time now. Thanks for all the ideas. I'll get back to it tonight or tomorrow.

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    Posts crossing again.
    and the proportion of 0/1 to 1/1s inside the X. The first issue can be ignored. Can you tally up 0/1s and 1/1s in the X chr from the vcfs?
    I hope you got my first post today, which answers that question. Unfortunately, I am just out of time now. Thanks for all the ideas. I'll get back to it tonight or tomorrow.

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    Ysearcher, if you want realign male chrX in haploid mode (non diploid as autosoma) you can do it with bcftools call that has an specific parameter to call genotypes in one allele.
    About annotated VCFs. Ensembl has updated VEP realise 96 with all UCSC/RefSeq/Ensembl detected variants. You can upload your WES vcfs directly to their site and run.
    http://grch37.ensembl.org/Homo_sapie...-5373550;to=50

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  11. #18
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    Thanks much for the links, Miqui. When I first got these whole exomes, I had a sponsored account to Quiagen Ingenuity Variant Analysis, compliments of the Personal Genome Project. It was all very new to me, so I floundered around with the various analysis tools, but still managed to pull up some interesting variants. It has taken me several years to replicate that, but I recognize some of the analysis tools from your link. The VCF file for these whole exomes is rather small, about 12.5 MB, so I presume I can upload the entire exome? I'll play with it a little and get back to the forum. I have discovered a handful of potentially novel coding variants, so I'll see if I can plug those in right away and get a VEP response. Sounds like a good plan.

    EDIT: Just running a few novel & extremely rare variants - this is great, just what I have been looking for! It has already confirmed three proteins coding effects that I did the hard way. Thanks a lot!
    Last edited by Ysearcher; 06-16-2019 at 08:13 PM.

  12. #19
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    I've been plotting the heterozygosity ratios of my various VCFs. For westerners I believe a 1.5x ratio of Het:Hom is normal across the genome. I've more or less confirmed this for myself.

    https://academic.oup.com/bioinformat.../3/318/2366248

    When I turn to the X though I get a variety of ratios. I looked at some of the Genome in a Bottle benchmark VCFs and they showed a very low non-zero het count on the X. eg HG002 GATK HaplotypeCaller benchmark had 4421 het calls v 90740 hom calls. A lot of their output doesn't even cover the X but I emailed them and got the links to some that do:

    "We haven't yet made benchmark calls on X and Y for the Ashkenazi and Chinese trios, since they are haploid in the male. We do have individual callsets (not benchmarks) that include X and Y in the inputvcfandbeds directory, such as
    ftp://ftp-trace.ncbi.nlm.nih.gov/gia...utvcfsandbeds/
    as well as bam files that contain X in under:
    ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/


    Alternatively, we have benchmark calls for X in HG001/NA12878 (a female) under
    ftp://ftp-trace.ncbi.nlm.nih.gov/gia...01/NISTv3.3.2/
    "
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