support of DMD sequencing
Bernabini Sara
Hi,
we are contacting you to have some information that can help us to better understand an issue we have encountered analyzing NGS data of DMD gene (NM_004006.2).
In particular, using UCSC database the position g.31893307 correspond to the first base of the intron 48 (c.7095+1), whereas using the NCBI database the same genomic position correspond to the first base of the last codon of exon 48 (c.7096).
We use the Alamut Software for variant interpretation and initially we asked to the Interactive Biosoftware Staff. You can see their answer below.
Can you please help us to understand this issue?
Thank you very much,
Kind regards,
Sara
Sara Bernabini
SOD Diagnostica Genetica
AOU Careggi
Tel +39 055 7949036
Fax +39 055 7949686
Da: Support IBS [mailto:sup...@interactive-biosoftware.com]
Inviato: lunedì 13 giugno 2016 17:39
A: Bernabini Sara
Oggetto: [1940] Alamut software assistance
Dear Sara,
We have looked carefully at your issue and we have found there is a difference in the way in which Alamut Visual and UCSC browser build transcript structure regarding DMD gene.
Our list of exons coordinates is determined using NCBI Refseq data and the NCBI Splign result tool. You can take a look at the Splign results for NM_0446.2 transcript alignment vs GRCh37 (hg19) build attached to this email : 1940-1.png and 1940-2.png. You can notice that the Lysine (with genomic coordinates from 31893305 to 31893307) is a part of exon 48.
If you take a look at the UCSC results the mapping of NM_004006 on the genome sequence on UCSC through the link https://genome.ucsc.edu/cgi-bin/hgc?hgsid=498774721_Q6QaJoouGv4dyI0D2SzGnHhoaBgq&g=htcCdnaAliInWindow&i=NM_004006&c=chrX&l=31119227&r=33211556&o=31119227&aliTable=refSeqAli&table=refGene (and the attached picture 1940-3.png), they consider this amino acid as a part of intron 48
According to us, Alamut Visual definition is more appropriate. In our build, we conserve the invariant AG sequence which is located at the splice acceptor site at the 3' end.
We also checked ENSEMBL alignment (http://grch37.ensembl.org/Homo_sapiens/Transcript/Exons?db=core;g=ENSG00000198947;r=X:31115794-33357558;t=ENST00000357033 and the attached picture 1940-4.png) and we got the same result as Alamut Visual.
You can also submit your findings to UCSC to understand better this issue.
At your beck and call for any other question.
Best regards,
Viet-Dung
Viet-Dung Tran
Bioinformatics Scientist
Interactive Biosoftware
T: +33 278 775 119
141 boulevard de l’Yser • Rouen • 76000 • France
vt...@interactive-biosoftware.com
www.interactive-biosoftware.com
From: Bernabini Sara [mailto:berna...@aou-careggi.toscana.it]
Sent: vendredi 10 juin 2016 15:11
To: Support IBS <sup...@interactive-biosoftware.com>
Subject: Alamut software assistance
Dear Viet-Dung, thanks for your replay.
Our Refseq Databas is UCSC (hg19).
Awaiting your reply about point number one.
Thank you, best regards
Sara Bernabini
Da: Support IBS [mailto:sup...@interactive-biosoftware.com]
Inviato: venerdì 20 maggio 2016 12:15
A: Bernabini Sara
Oggetto: [1940] Alamut software assistance
Dear Sara,
Could you please give us some more descriptive reference about the Refseq Database to better understand the issue (For instance your genome viewer to help us to reproduce the issue in which the genomic position 31893307 on chrX (hg19) the RefSeq database identifies the first base of the intron 48 of the DMD gene).
For our part, we use Splign program to align Transcript from RefSeq record on assembly build.
About the splicing prediction interpretation, you must consider the mutation context and all prediction data.
We recommend you these two articles of Dr. Houdayer et al which can help you for the splicing prediction interpretation:
- In silico prediction of splice-affecting nucleotide variants in In Silico Tools for Gene Discovery, Springer, 2011.
- Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.
Hum Mutat. 2012 Aug;33(8):1228-38.
Please find below the documentation link which describes the splicing module:
http://www.interactive-biosoftware.com/doc/alamut-visual/2.7/splicing.html
To reveal differences between wild-type and mutated splicing predictions scores, click on the 'Highlight Differences' button on ‘Splicing Window’ (please see attached picture). Unchanged scores get dimmed, while score numbers are displayed beside those that differ. This difference can reveal new splicing site, or inversely, existing site is broken.
At your beck and call for any other question.
Best regards,
Viet-Dung
De : Bernabini Sara [mailto:berna...@aou-careggi.toscana.it]
Envoyé : jeudi 19 mai 2016 15:30
À : Madina Nataeva
Objet : Alamut software assistance
Importance : Haute
Dear Madina,
I am writing to you because I need your kind assistance to resolve some difficulties I have encountered using Alamut software to interpret NGS variants:
1) We noticed a difference between the RefSeq database and Alamut in the definition of the coding sequence of the DMD gene. In particular, at the genomic position 31893307 on chrX (hg19) the RefSeq database identifies the first base of the intron 48 of the DMD gene (NM_004006:c.7095+1A>C), whereas Alamut identifies at the same position the last codon of exon 48 (NM_004006:c.7096). Can you please help me to understand these differences? The interpretation of the variant could be quite different (splicing vs non synonymous aa change)…
2) Can you please give me some instruction useful to better understand and use the Alamut tool for splicing prediction? Can you please share with me your opinion on the significance of the splicing prediction you can find in the attached ppt files? They are synonymous variants in the DMD gene but their frequencies are really low (below 0.02).
Thank you in advance,
Sara
Sara Bernabini
SOD Diagnostica Genetica
AOU Careggi
Tel +39 055 7949036
Fax +39 055 7949686
Matthew Speir
Thank you for your question about the RefSeq Genes track in the UCSC Genome Browser.
Please see this answer to a previous mailing list question as to why the coordinates for RefSeq transcripts in the UCSC Genome Browser may differ from those at NCBI or other sources: https://groups.google.com/a/soe.ucsc.edu/d/msg/genome/kJh3YJCiCDs/PEGTRqZdMwAJ.
We are working to create a track here at UCSC that contains coordinates and alignments directly from RefSeq so that what is displayed in our browser matches what's seen at RefSeq. Unfortunately, though, I do not have an estimate on when this track may be released.
I hope this is helpful. If you have any further questions, please reply to gen...@soe.ucsc.edu. All messages sent to that address are archived on a publicly-accessible Google Groups forum. If your question includes sensitive data, you may send it instead to genom...@soe.ucsc.edu.
Matthew Speir
UCSC Genome Bioinformatics Group
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