Human Genome Variants Analysis. Marin Vargas, Sergio Paul

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1 Human Genme Variants Analysis Marin Vargas, Sergi Paul 2013

2 Why make predictive diagnsis f a genetic disease using the whle genme? Genme sequencing is the nly way t get all genetic infrmatin. The cst f whle genme sequencing is drastically decreased. The Next Generatin Sequencing (NGS) technlgy has been remarkably successful in finding the causes f Mendelian and rare diseases. With persnalized medicine we can get targeted therapies.

3 SNV might cause a genetic disrder SNV: Single Nucletide Variant Sickle cell anemia SNP: Single Nucletide Plimrfism If a SNV is present in at least 1% f ppulatin SNP. If a SNV isn t present in at least 1% f ppulatin Mutatin.

4 Frm Genme Variants t patient s genetic disease diagnsis DNA Extractin DNA Sequencing (Genme r Exme) Genme reference Variants Calling (Biinfrmatics Prcess) Variants Analysis (Biinfrmatics Prcess)

5 DNA extractin The Centr Genmica Funzinale (CGF) f Prf. Dellednne has available a sequencer Illumina Hiseq 1000 with Next Generatin Sequencing (NGS) technlgy.

6 DNA sequencing Next Generatin Sequencing (NGS) (reads) Lad int sequencer T be reliable enugh t sequence the entire genme high cverage is required. Human Genme (~ 3 Gb) needs mre than 90 Gb. Human Exme (~ 135 Mb) needs mre than 4 Gb.

7 DNA sequencing result The result f NGS is a file in FASTQ frmat cntaining all paired-end reads (length every single read ~ 100) f the Exme/Genme sequenced. (Sequence)

8 Variant Calling Raw NGS reads quality filtering. Alignment t reference genme (BWA). Variants calling (GATK). Example VCF file:

9 Aligment t reference genme by BWA Burrws- Wheeler Aligner

10 Variant Calling by GATK

11 Variant Analysis Genme ~ 5,000,000 variants Exme ~ 200,000 variants The variant analysis is based n different filtering levels: Allele frequency (Cmmn Variants). Exnic and splice regins and variants are nt synnymus (N bvius effect n prtein). Prbability that the mutatin is harmful and that the cnservatin sites are prbably very imprtant fr the prtein functinality (Bilgical interpretatin). Hmzygus and heterzygus mutatins are different. The expected result are a few bilgically relevant variants.

12 Variant Analysis Sftware Human Genme Interpreter Free academic sftware: Functinal anntatin f genetic variants frm high-thrughput sequencing data Paid sftware:

13 Variant Analysis - Case f study 1 We analyzed the exme f a 20-year-ld man with Alagille syndrme and Acute Lymphblastic Leukemia. Mre than 280,000 variants were analyzed using KNOME sftware. We fund putative damaging mutatins in genes such as PAX5 (R38H) and NOTCH1 (K1821N) which might be strngly related t the bserved disease. The publicatin abut this wrk is nging.

14 Variant Analysis - Case f study 2 We are analyzed tw families: The first family had a phentype f a neurmuscular disease tgether an inflammatry disease. Their exmes have been analyzed using ANNOVAR. We fund a mutatin in the sn s gene NEB (I6512T) crrelated with the Nemaline mypathy disease. The secnd family had Palmplantar keratderma (PK) disease and a Charct marie tth type 2 (CMT) disease. Their exmes have been analyzed using ANNOVAR. We fund a mutatin in the gene KRT1 (F200L) fr these nes with PK disease and fund a mutatin in the gene MPZ (S44F) fr these nes with CMT disease, n mutatins were fund in these genes fr healthy patient.

15 del 30/10/2013 Alla ricerca dei geni respnsabili delle malattie IL PROGETTO DOSE STUDIA LE DIFFERENZE TRA LE QUANTITÀ MINORE O MAGGIORE DEI GENI E LE CONSEGUENZE CHE QUESTO HA PER LA SALUTE L'era infrmatica che stiam vivend ha frnit gli strumenti affinché la bilgia pssa prdurre un numer enrme di sequenze di DNA di mlte specie diverse. La tecnlgia mderna ha res il sequenziament del DNA più semplice, men csts e più affidabile, cn enrmi benefici per la diagnsi e la cura delle malattie. In passat la difficltà stava nel raccgliere dati genetici, ggi la sfida è dare lr un sens. "Stiam assumend un apprcci evlutiv per dare un sens alle sequenze di DNA, ciò significa che esaminiam il md in cui i geni si sn evluti, per capire megli cme funzinan. Cn il su prgett DOSE ("Dsage sensitive genes in evlutin and disease"), finanziat dall'ue, McLysaght sta studiand le differenze tra le dsi dei geni, vver, pssedere una quantità minre maggire del gene, e le cnseguenze che quest ha per la salute. "Le variazini delle quantità di un gene tra individui - variazini di dse - sn una scperta relativamente recente e a vlte sn implicate nella malattia", spiega McLysaght. In termini semplici, la dttressa ha adttat un apprcci evlutiv per scprire quali variazini di dse sn accettabili e quali sn prbabilmente cinvlti nelle malattie umane. Osservand l'evluzine pssiam capire le variazini accettabili e inaccettabili del DNA", spiega McLysaght. "I cambiamenti del DNA che si sn rivelati inaccettabili durante l'evluzine sn prbabilmente gli stessi di quelli che ggi causan le malattie Genme-wide deserts fr cpy number variatin in vertebrates (articl pubblicat)