Next Generation Sequencing in Science and Medicine

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Next Generation Sequencing in Science and Medicine,The Presidential Commissionfor the Study of Bioethical Issues,28 February 2011,Washington D.C.,University of North Carolina at Chapel Hill,1,DNA Has Only Two Jobs,It serves as a store of information,Ensuring that information is passed on to each new cell upon division (and the next generation),It directs the synthesis of proteins,Which are necessary to carry out the functions of a living organism,2,DNAs Structure Explains How it Accomplishes Both Jobs,Thymine (T),Guanine (G),A,T,G,C,G,T,C,A,Adenine (A),Cytosine (C),Always binds to,Always binds to,As a double helix, each DNA molecule contains a copy of itself,DNA Serves as a Store of Information,3,DNAs Structure Explains How it Accomplishes Both Jobs,It is the,order of the bases,that provides the instructions for protein synthesis,One stretch of DNA directs the synthesis of one type of protein, another stretch directs the synthesis of another type of protein,DNA Directs the Synthesis of Proteins,T,G,C,A,4,We call a stretch of DNA that directs the synthesis of one particular protein a,Gene,Gene #1,Gene #2,Gene #3,5,DNA Sequencing,You have 3 billion bases arrayed in a unique order, with 20,000 genes that direct the synthesis of all the proteins that comprise you,“Sequencing” DNA is simply the elucidation of the order of the bases in an organisms DNA strand,The unique order of your bases greatly influences your health, e.g.,What disease you are more - or less - prone to,How you will react to different medications,A human “genome” is about 6 feet of DNA,And each of your cells contains two copies of your genome,6,A Human DNA Sequence,1/1,000,000,th,of the Human Genome,Interspersed with genes,And “polymorphisms” which differ between people,Are sometimes important, influencing traits,or medically important characteristics,atcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatctactagaagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattggaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtagccctacgtaccgtcggtactggtaacgtgaggtcaggttgttcaactcatccaggattagatccgtagatcgtaggaaatatctcggataattaacagatacacacccttagaccatttaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctgggaaaaattgggccctacgtgtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgacgtttccaggctacacacacactgacagatagacagattcaaattcagtcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcatatatccaggaaaatccctgggaaaaattggctacgtaccgtattaactaggatctccgatggtacccattaagacacccaaaataggtaacaggtagacatattgatacccatagaggatagatttaggacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccagctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacgggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctgggaaaaattgggccctacgtgtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcgccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcacacacacactgacagagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgcagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgagagtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctggactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggca,7,Sequencing DNA,Early techniques were developed in the 1970s,A variety of approaches now exist,The biggest limitation to sequencing is that the genome is big,So carrying out these reactions for an entire genome is slow and expensive,8,Next Generation Sequencing,Takes advantage of miniaturization to engage in massively parallel analysis,Essentially carrying out millions of sequencing reactions simultaneously in each of 10 million tiny wells,Sophisticated computer analysis of huge amounts of information allows “assembly of a given sequence,9,$795 in 1977,(=$2,800 in current $),Accelerating Technology & Plummeting Cost,Next Generation Sequencing,10,Genomics,Long Term Promise,An avalanche of Genome Wide Association Studies (GWAS) in common diseases,WGS is now a practical reality,Will shed light on the genetic underpinnings of every disease imaginable,Ultimately transforming Medical Science,11,Medical Science = Medical Practice,Medical Science is the indispensible foundation of Medical Practice,But medical practice is far more complex,More variables,Individual values matter and they differ,Theory alone is insufficient to guide practice,Time-line for translation is long,Successful translation into practice is,not guaranteed by scientific understanding,See sickle cell disease,It,s far more expensive,The stakes are much higher in Medical Practice,Because the power to harm is real and potent,12,Where Does the Clinical Promise of Genomics Lie?,Probably not in risk assessment for common disease,Common diseases have many factors, of which genetics is only one,The bulk of any individuals genetic risk for common disease is modest,Thus assessing genetic risk only “nudges” that risk slightly,Few data to suggest that knowledge of ones genomic status for common disease is useful or is effective in changing behavior,Genetics,Diet,Exercise,Smoking,Diabetes,Hypertension,Probably not in simply assessing risk,13,Whats The Right Nail For Sequencing Technology?,As a diagnostic tool in enigmatic patients,As a public health tool to identify those apparently well individuals with dramatically increased risk of preventable disease,14,Next Generation Sequencing as a Clinical Diagnostic Tool,47 yo female with sudden cardiac arrest,Resuscitated successfully,EKG reveals “Long QT Syndrome”,High risk for sudden death,Dozens of genes implicated,Application of NGS to detect mutation,Thereby guiding patients treatment and prevention of death in family members,?,?,?,?,?,15,Next Generation Sequencing as a Public Health Tool,0.25% of US women (375,000) carry a mutation in,BRCA1/2,At very high risk of breast and ovarian cancer,85% lifetime breast cancer risk,25-50% lifetime ovarian cancer cancer,Knowledge of risk allows prevention,Currently we only can identify such women once several family members have developed cancer,NGS allows population screening for high risk preventable disorders,Cancer predisposition, cardiac disease, etc.,1-2% of population carry such mutations,3-6 million individuals in the US with preventable disorders if identified,16,Challenges to Harnessing NGS in Clinical Medicine & Public Health,Accuracy,99.99% accuracy x 3 billion nucleotides,= 300,000 errors,per patient,Interpretation of the variants we find,Storage and access in the medical record,Education of patients and public,Issues of consent and reporting,Education of providers,17,Incidental Information,Upon WGS we discover many things we werent looking for, which we can do nothing about,Some are trivial or indeed beneficial,But some are problematic,And we will occasionally discover lethal, untreatable late onset conditions,Some wish to know such information; others do not,We must grapple with how to inform patients about such information,protect patients from harm but also allow individual autonomy and choice,Low risk,incidental information,Medium risk incidental information,High risk,incidental information,PGx variants and common risk SNPs with no proven clinical utility,APOE,Carrier status for severe recessive diseases,Huntington disease,Prion diseases,etc.,18,In the Clinical Arena, Genetic Information Presents Few,Qualitatively,New Challenges,Genetic tests affect others,Infectious Disease,Provide probabilistic information to the asymptomatic,Cholesterol,Our genome cannot be changed,Nor can much of what we discover medically,Insurance discrimination,Actually better for genetics than the rest of medicine,Unexpected results, FPs/FNs,Are a routine part of all clinical care,DNA is,“,uniquely identifiable,”,Zip code + DOB + spouse,s first name,ATGGTCCTATGGATCTCTTTAAAGGGCCTTAGTACTTACCGTAACTTTAGCCGGTAGCTTAATCGTGCCTAGGTCGATTGCCTAGGCTTAGCTAGCTTGG,Jim,s Whole Genome Sequence!,Eschewing Genetic Exceptionalism,19,Challenges to Realizing Genomic Medicine,Creation of a centralized, evidence-based, iterative process to define clinically significant genomic findings,Thorough health-oriented phenotypic annotation of variants,Enabling realistic shared decision making among a range of providers, technology interfaces and patients,Understanding the ethical dimensions, patient preferences & values regarding returning incidental results,Maintaining a sober focus on evidence,Life is short, the art long, opportunity fleeting, experience delusive, judgment difficult,20,21,22,23,A Human DNA Sequence,1/1,000,000,th,of the Human Genome,Interspersed with genes,And “polymorphisms” which differ between people,Are sometimes important, influencing traits,or medically important characteristics,atcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatctactagaagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattggaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtagccctacgtaccgtcggtactggtaacgtgaggtcaggttgttcaactcatccagga/taaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctgggaaaaattgggccctacgtgtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgacgtttccaggctacacacacactgacagatagacagattcaaattcagtcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcatatatccaggaaaatccctgggaaaaattggctacgtaccgta/gacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccagctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacgggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctgggaaaaattgggccctacgtgtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggc/gccttgaatcttggcagtcgtaacgtactagaatatatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgacagatagacagattgtcgtgttatvtgacttggaactgtaggcacacacacactgacagagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgaggtcaggttgttcaactcatccaggaaaatccctgggaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtacgtttccaggctacacacacactgcagatagacagattgtcgtgttatvtgacttggaactgtaggcccttgaatcttggcagtcgtaacgtacgtacggtactggtaacgtgagagtcaggttgttcaactcatcgtgactgattaccaggatcctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgagaaaaattgggccctacgtaccgtaacgttgcaaattcagtcggtctagcggatcctactgacctgacgtacgtaatgcagtggtcaggttgttcaactcgatgactagaatatatccaggaaaatccctggactcat