Bou De Pieri, Francesc

Universitat Autònoma de Barcelona. Facultat de Biociències

Bou de Pieri, Francesc

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WHOLE EXOME SEQUENCINGBACKGROUD1st. generation sequencingIrreversibleterminationEx: Sanger3rd. generation sequencingSingle DNA moleculeEx: Heliscope, SMRT, NanoporeSequencingplatformsExome sequencing• Targeted sequencing enrichment focused on all protein-coding subsequences.• Hybrid capture (microarray), solution capture (proves).• Cost-effective, reproducible and robust strategy for the identification of variants causing protein-coding changes in individual human genomes.Neural tube defects• An opening in the spinal cord or brain that occurs very early in human development as a result of the failure in neural tube closure (1/1,000 live births).• Multifactorialinheritance.• Complex disease model.2nd. generation sequencingSynthesis,ligation,hybridizationEx: 454,HiSeq, SOLiD1stNext generation2nd 3rdSanger 454 HiSeq SOLiD HeliscopeMechanismDideoxy terminationPyrosequencingSeq. by synthesisSeq. by ligationSingle molecule seq.Read length 400-900bp 400-600bp 100-200bp 50-100bp 25-55bpAccuracy 99.999% 99.5% 98.5% 99.94% >99%Output data/run1.9-84Kb 0.7Gb 600Gb 120Gb 50GbTime/run 20min-3h 24h 3-10 days 7-14 days 8 daysCost/Mb S 2400 S 10 S 0.07 S 0.13 >2nd. gen.+High qualityLong readsLong readsFastHigh throughputHigh accuracy (twice)Easy and fast preparation-High cost High costShort reads Short readsShort readsAdvantages DisadvantagesLong readsExcellent accuracyHigh qualityHigh costLimited throughputTime consumingGel-based size-fractionationAdvantages DisadvantagesHigh throughputIncreased speedProduce over 100 more dataReduced costFewer preparatory stepsNo gel-based size-fractionationShort readsPCR neededHigher error ratesHigh redundancy requiredComputational demandsComprehensive data analysisAdvantages DisadvantagesLess initial DNANo PCRBetter accuracyLower throughputShort readsFinal Degree ProjectFrancesc Bou De Pieri, Genetics Degree, UABOBJECTIVES RESULTS AND DISCUSSIONMATERIALS AND METHODS• Carry out a comparative analysis between genome sequencing and whole exome sequencing techniques.• Demonstrate the importance and the applications of whole exome sequencing.• Use whole exome sequencing as an approach to identify genetic factors causing complex diseases, such as neural tube defects.Bibliographic researchPapers published in PubMed.Words used: “Next Generation Sequencing”, “Whole Genome Sequencing”, “Whole Exome Sequencing”, “Comparison” and “Application”.WES-Targeted captureSureSelect 50Mb human exome kit (Agilent)635k provesWhole genome sequencing (WGS)• Results can be very sensitive to false positives and false negatives• Needs a validation process• Relies on the controls or the reference genome used to map the reads obtained from the sequencing processWhole exome sequencing (WES)• Make sequencing feasible for a huge variety of laboratories in terms of costs and infrastructures• Protein coding genes constitute only 1% of the human genome but harbor 85% of the mutations with large effects on disease traits• Limited view (coding regions)• WES coding SNP detection has as sensitivity as WGS• Efficient approach to identify genes causing mendelian disorders.Low throughput Low throughput Low throughputAdvantages DisadvantagesWide availabilityImproved cost efficiencyDetects large structural variantsDetects coding and non-coding variantsProduces 100 times more dataHigh costShort read lengthLow interpretabilityHigh computational needs (filtering, storage, software and hardware)Advantages DisadvantagesLower costLess raw sequence neededAllows bigger sample sizeIncreased sequence coverageDetects coding variantsSensitive and specific identificationDoes not detect structural variantsNot all targets are captured (80%)Difficult to capture ↑GC% regionsLimited viewSamplesCentral nervous system tissue from diagnosed fetuses.• Normal karyotype by G-banding• No CNVs clearly linked to the phenotypeCONCLUSIONSBIBLIOGRAPHY•• High efficiency -SequencingSOLiD 5500 sequencing (Applied Biosystems)• High accuracy-MappingSOLiD LifeScope software (Life Technologies)• Iterative mapping approachHuman reference genome hg19 (UCSC)Variant analysisAll variants observed should be filtred by:• Sample recurrence (present in 2 or +)• Presence of frameshift and nonsense mutations or predicted pathogenicity (Condel)• Conservation (PhyloP value)ValidationSanger sequencing of PCR amplicons from genomic DNA:• Design a pair of primers for each variation• Do the PCR tuning• Put the PCR with the samples• Check the PCRs with agarose gel• Purify the DNA from PCR• Do the sequencing PCR and the precipitation of DNA• Analyze the sequences obtained from Sanger sequencingApplication of WES in a complex disease• WGS and WES, shows that both approaches can be used to identify genetic factors causing human diseases• WES has been a very useful and efficient approach to identify the genetic causes of mendelian disorders but has a limited view.• WES can be also used to identify the genetic causes of multifactorialdisorders.Despite the true advantages that WES offers now, in the future, WGS is predicted to be more economical than WES because the capture process is skipped entirely. Meanwhile, WES is considered the best approach because it provides most of the benefits of WGS but with lower costs and higher clinical interpretability.1- Lin, B., Wang, J. & Cheng, Y. Recent Patents and Advances in the Next-Generation Sequencing Technologies. NIH Public Access 2008, 60–67 (2008).2- Majewski, J., Schwartzentruber, J., Lalonde, E., Montpetit, A. & Jabado, N. What can exome sequencing do for you? J Med Genet. 48, 580–589 (2011).3- Gilissen, C., Hoischen, A., Brunner, H. G. & Veltman, J. a. Unlocking Mendelian disease using exome sequencing. Genome Biol. 12, 228 (2011).4- De Marco, P., Merello, E., Mascelli, S. & Capra, V. Current perspectives on the genetic causes of neural tube defects. Neurogenetics. 7, 201–221 (2006).SamplesCaptureSequencing MappingFilterValidation Candidate genes list (20-30)Genetic factors of complex diseasesBibliographyPCPPlanar Cell PolarityFolate

Whole exome sequencing

http://ddd.uab.cat/pub/tfg/2014/126260/TFG_francescboudepieri.pdf

Whole exome sequencing

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