problem set 6 questions from class 2018 PDF

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Problem set 6 ...

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& Problem&Set&is&due&in&class&before&11:10am&on&Tuesday,&March&6,&2018.& & Instructions* For*this*problem*set*you*are*to*work*in*groups*of*3*students*to*find*solutions*to*the*problems.*After* working*with*your*group,*write*and*submit*your*own*solutions*as*part*of*your*problem*set.*You*may* (but*are*not*required*to)*submit*identical*solutions.*Follow*guidelines*below*for*group*work*and* submit*the*evaluation*form*at*the*end*along*with*your*completed*problem*set.*& & Guidelines:& Work*in*your*assigned*group.*Don’t*switch*groups*unless*you*have*been*given*permission*from*an* instructor.& Work*together*as*a*group*on*each*problem*rather*than*dividing*up*the*problems*and*swapping* answers.*& • All*group*members*should*do*an*equal*amount*of*work.*& • Don’t&take&over&the&discussion&and&don’t&let&others&take&over.&& • Make&sure&everyone&is&able&to&voice&their&ideas&and&questions.&& • Listen&effectively&and&don’t&be&critical&of&others.& • Speak&up&if&you&don’t&understand&what&another&group&member&is&saying.&It&is&important&to&let& people&know&when&what&they&are&saying&is&unclear.& • Be&patient&if&you&understand&something&others&don’t.&Help&each&other&understand&all&of&the& concepts.&The&best&way&to&learn&something&is&to&try&to&explain&it&to&another&person.& & Names&of&group&members:&_________________________&&&&&&&&&_________________________& & Evaluation&form:& & (1) Self-evaluation:&Evaluate&your&contribution&to&the&group.& & & & (2) Team&evaluation:&Evaluate&each&group&member’s&contribution.& & & & & (3) Group&dynamics:&Describe&how&well&the&group&worked&together.& & & & & “On&my&honor,&I&have&neither&given&nor&received&inappropriate&assistance&in&the&taking&of&this&exam&or& problem&set.”& Sign&here:&___________________________________________& & Upload*your*completed*problem*set*as*a*single*pdf*file*in*bcourses*under*Assignments/s18*_c148_PS6.*

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& 1. For&each&of&the&scenarios&below,&indicate&which&of&the&following&techniques&you&would&use& to&answer&the&research&question:&licon&sequencing,&shotgun&metagenomics,&or& comparative&genomics.&Explain&your&choice&and&briefly&describe&how&you&would&perform& the&experiment,&including&the&sample(s),&experimental&design&(don’t&include&details&about& how&sequencing&is&done,&just&say&Illumina&sequence&or&Sanger&sequence),&and&analysis&(e.g.,& compare&data&from&X&and&Y,&explain&how&you&will&interpret&the&data).&& & a) You&are&curious&to&see&how&taking&antibiotics&affects&the&gutµbiome&and&have& recruited&50&volunteers&who&have&been&prescribed&the&same&14-day&course&of& antibiotics.&You&are&specifically&interested&in&the&following&question:&Which&bacterial&taxa& in&the&gut&are&most&affected&by&the&introduction&of&antibiotics?&[6&points]& & Sample:&&& & & Experimental&design:&& & & Analysis:&& & & & & b) You&are&a&soil&scientist&and&you&have&used&FISH&to&observe&the&spatial&arrangement&of& bacteria&in&several&soil&samples.&You¬ice&that&two&types&of&bacteria&always&occur& together&(i.e.&there&is&no&sample&where&only&one&of&the&species&appear).&You&are&able&to& isolate&single&cells&of&each&bacterium&but&have¬&been&able&to&culture&them.&This&leads& you&to&the&following&question:&Do&these&two&species&share&necessary&metabolites&in& order&to&survive?&[6&points]& & Sample:&& & Experimental&design:&& & Analysis:&& & & & & & & &

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& c) In&some&patients&prescribed&the&heart&medication&Digitalis,&the&active&ingredient&digoxin& is&reduced&to&an&inactive&form&by&a&gut&bacterium.&Scientists&have&identified&the&genes& responsible&for&digoxin&reduction&in&the&cgr&operon.&You&are&curious&about&where&the&cgr& genes&are&found&in&nature.&Digoxin&is&naturally&produced&in&the&leaves&of&the&plant& Digitalis*lanata.&You&have&acquired&a&leaf&sample&from&a&D.*lanata&plant&and&want&to& answer&the&following&question:&Are&the&cgr&genes&present&inµbes&that&live&in& association&with&D.*lanata*leaves?&[6&points]& & Sample:&& & Experimental&design:&& & & Analysis:&& & & & 2. For&each&component&of&a&genome&below,&give&at&least&one&reason&that&it&might&be& challenging&to&assemble&and&bin&complete&genomes&containing&the&given&element&using& shotgun&metagenomics:&[3&points&each]& & a) Plasmids:&& & & & & & & & b) Transposons&and&large&repeats:&& & & & & & & & c) Viral&genes&(prophage):&& & & & & &

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& 3.&Many&fungi,&including&the&common&member&of&the&human&gut&and&opportunistic&pathogen& Candida*albicans,&have&multiple&linear&chromosomes&and&are&diploid&(have&two&copies&of&each& chromosome).& & & & & & & & & & & In&the&figure&above,&read&coverage&is&represented&across&chromosomes&1,&6,&and&7&of&21& different&clinical&isolates&of&Candida*albicans.&Each&clinical&isolate&was&cultured&and&sequenced& using&Illumina&sequencing.&The&read&coverage&is&normalized&to&the&coverage&of&the&diploid& reference&strain,&SC3514.&On&chromosome&1,&strain&P94015&has&a®ion&with&double&the& coverage&of&the&rest&of&its&genome&and&the&authors&have&found&it&is&tetraploid&in&this®ion& (contains&four&copies&of&this§ion&of&the&chromosome)&rather&than&diploid.&Such&instances& where&an&abnormal&number&of&chromosomes&are&present&are&referred&to&as&aneuploidy.& & a) What&can&you&conclude&about&the©&number&of&following&chromosomes:& & 1.) Chromosome&7&in&strain&19F&[3&points]& & & & & 2.) Chromosome&1&in&P57072&[3&points]& & & & & b) Strains&P94015,&P60002,&L26,&and&19F&grew&much&slower&in&all&growth&media&tested& compared&to&all&other&isolated&strains.&What&does&this&possibly&tell&you&about&the&effect& of&the&observed&aneuploidy&events&on&the&fitness&of&Candida*albicans&in&laboratory& conditions?&What&about&in&in*vivo&conditions?&[4&points]& & & & & & & &

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& c) A&colleague&sequences&a&new&Candida*albicans&genome&using&single-cell&genomics& methods.&They&observe&extremely&variable&read&coverage&across&their&genome& compared&to&the&reference&strain&SC3514.&They&conclude&this&variability&is&due&to& aneuploidy.&Do&you&agree&with&their&conclusion?&Why&or&why¬?&[4&points]& & & & & & & & & 4)&You&are&interested&in&studying&nitrogen&cycling&by&bacteria&in&the&Monterey&Bay.&You&decide& the&approach&you&would&like&to&take&is&to&perform&shotgun&metagenomics&on&ocean&water&from& the&bay,&reconstruct&genomes&for&the&organisms&present,&and&then&analyze&the&metabolic& potential&of&the&reconstructed&genomes.&Two&of&the&complete&genomes&you&recover,&named& Thermus*thermophilus*JL-18&and&Riemerella*anatipestifer*CH3,&are&of&particular&interest&to&you& because&they&may&play&a&role&in&denitrification&(both&of&these&genomes&in&reality&were& sequenced&in&isolate&but&imagine&they&were&sequenced&using&shotgun&metagenomics).&& & The&links&below&lead&to&the&KEGG&database,&a&useful&tool&for&examining&the&metabolic&potential& of&organisms&of&interest.&Specifically,&the&links&lead&to&a&summary&of&nitrogen&metabolism&for& both&Thermus*thermophilus*JL-18&and&Riemerella*anatipestifer*CH3.&Green&boxes&indicate& enzyme&annotations&that&are&present&within&the&genome&whereas&white&boxes&indicate& annotations&that&are¬&present.&Use&the&KEGG&database&to&answer&the&following&questions.&& & Riemerella*anatipestifer*CH3* http://www.genome.jp/keggbin/show_pathway?org_name=rat&mapno=00910&mapscale=&show_description=hide& Thermus*Thermophilus*JL-18* http://www.genome.jp/keggbin/show_pathway?org_name=ttl&mapno=00910&mapscale=&show_description=hide& & a)&Does&Riemerella*anatipestifer*CH3&have&the&full&denitrification&pathway?&If¬,&what&portion& can&it&perform?&[3&points]& & & b)&Does&Thermus*thermophilus&JL-18&have&the&full&denitrification&pathway?&If¬,&what&portion& can&it&perform?&[3&points]& & & &

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& c)&Does&the&community&as&a&whole&have&the&potential&to&perform&the&full&denitrification& pathway?&Why&or&why¬?&[4&points]& & & & & & & & d)&After&isolating&the&two&bacteria&you&find&that&Riemeralla*anatipestifer*CH3&is&capable&of& performing&dissimilatory&nitrate&reduction&all&by&itself&without&the&help&of&any&other&bacteria.& Does&this&result&make&sense&with&the&enzymes&predicted&to&be&present&in&the&KEGG&map&for&this& organism?&If¬,&provide&a&reason&why&you&may&have&received&these&two&conflicting&results.&[4& points]& & & & & & & & & & & & e)&A&start-up&company,&ThioGone,&is&entirely&convinced&Thermus*thermophilus*JL-18&can&oxidize& Thiosulfate&to&Sulfate&based&on&your&genome&and&the&following&KEGG&map.&& http://www.genome.jp/kegg-bin/show_pathway?ttl00920& They&are&ready&to&move&forward&with&the&very&expensive&production&of&thiosulfate&oxidation& bioreactors&using&Thermus*thermophilus&JL-18&as&the&key&organism&but&first&ask&for&your&advice.& What&experiment&would&you&advise&the&company&to&do&before&starting&production?&[4&points]& & & & & & & & & & & & &

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& 5.&Answer&the&following&questions&about&genome&assembly&quality:& & a)&You&are&attempting&to&construct&a&reference&genome&for&a&new&family&of&choanoflagellates.& Based&on&previous&experiments,&you&know&the&genome&should&have&12&chromosomes&and&be& around&60&Mbp&in&length.&You&recently&received&your&Illumina&sequencing&reads&back&from&the& sequencing¢er&and&after&assembly,&receive&a&reconstructed&genome&that&has&187&scaffolds& and&is&62&Mbp&in&total&length.&&Would&this&genome&be&considered&a&Draft,&Complete,&or&Finished& genome?&Explain.&[3&points]& & & & & & & & b)&Describe&two&ways&in&which&you&could&improve&the&quality&of&this&genome&assembly.&[4& points]& & & & & & & & & c)&For&shotgun&metagenomics,&many&of&the&genomes&are&draft&quality.&An&additional&way&to& measure&genome&quality&is&to&assess&the&presence&or&absence&of&single©&orthologous&genes& (SCGs).&SCGs&are&genes&that&are&observed&to&be&present&in&a&single©&(and&only&one©)&in& every&complete&genome.&What&would&it&mean&if&a&genome&bin&was&missing&many&of&the& expected&SCGs?&[3&points]& & & & & & & d)&With&shotgun&metagenomics,&the&number&of&SCGs&present&in&more&than&one©&is&also&used& to&measure&the&quality&of&a&genome&bin.&What&would&a&genome&bin&with&many&SCGs&present&in& multiple&copies&be&indicative&of?&[3&points]& & & &

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& 6.*Vibrio*cholerae*is&an&aquatic&bacterium&that&causes&the&diarrheal&disease&cholera.&Despite&the& species’&name,&the&vast&majority&of&Vibrio*cholerae&strains&in&the&environment&cannot&cause& disease.&To&cause&cholera,&the&bacterium&must&produce&cholera&toxin&as&well&as&a&structure& called&the&toxin&co-regulated&pilus&(TCP).& & a) Are&the&genes&encoding&cholera&toxin&and&the&TCP&part&of&V.*cholerae’s&core&genome&or& its&accessory&genome?&Explain&why.&[3&points]& & & & & & b) Almost&all&V.*cholerae&isolates&that&cause&cholera&belong&to&the&O1&group,&a&lineage&of&V.* cholerae*that&does¬&produce&an&extracellular&capsule.&Some&V.*cholerae*isolates&in&the& O139&group,&a&different&lineage,&both&produce&capsule&and&cause&cholera.&The&genes& that&encode&capsule&as&well&as&the&genes&that&encode&cholera&toxin&and&the&TCP&are& located&within&genomic&islands&flanked&by&phage&genes&with&different&GC&content&from& the&rest&of&the&genome.&What&types&of&events&in&the&evolutionary&history&of&V.*cholerae& could&explain&why&only&some&V.*cholerae*strains&make&capsule&and&cause&cholera?&[4& points]& & & & & c) You&have&isolated&a&novel&V.*cholerae*isolate&from&San&Francisco&Bay.&Preliminary& experiments&show&that&the&isolate&produces&capsule&and&TCP,&and&does¬&produce& cholera&symptoms&in&a&mouse&model.&Describe&a&genomics&experiment&to&determine&(1)& whether&this&new&isolate&belongs&to&the&O1&lineage,&the&O139&lineage,&or&a&separate& lineage,&and&(2)&whether&it&is&capable&of&producing&cholera&toxin.&[10&points]& & & & & & & & & & & & & &

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PMB/MCB&c148&Spring&2018&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&ID:___________________&&NAME:&_____________________& Problem&Set&

& 7.&Suppose&you&have&isolated&aµbe&capable&of&efficiently&sequestering&CO2,&which&you&think& could&be&used&to&counteract&climate&change.&You&want&to&sequence&its&genome&with&paired-end& reads,&but&your&supervisor&is&unwilling&to&pay&the&extra&~$500&and&will&only&pay&for&single-end& reads.&What&could&you&say&to&argue&that&paired-end&reads&will&give&you&a&higher&quality&genome& sequence?&[4&points]& & & & & & & 8.&You&perform&both&16S&sequencing&and&deep&metagenomics&shotgun&sequencing&on&ocean& floor&sediment&samples&from&the&North&Atlantic.&To&your&surprise,&there&is&a&group&of&highly& abundant&organisms&in&your&shotgun&metagenomics&sample&that&were¬&detected&at&all&in&the& 16S&licon&sequencing&sample.&When&you&investigate&these&organisms&further,&you&realize& they&are&very&distantly&related&to&bacteria,&eukaryotes,&and&archaea&and&may&even&be&a&new& domain&of&life.&Their&gene&homologous&to&the&16S&has&also&been&shortened&compared&to& bacterial&and&archaeal&16S&sequences,&and&you&name&it&the&14S&rRNA&gene.& & a) Why&were&these&organisms¬&detected&with&16S&sequencing&but&still&detected&with& shotgun&metagenomics?&[3&points]& & & & & & & & & b) Would&it&be&possible&to&make&licon&sequencing&primers&that&would&work&for&the&14S& rRNA&gene&in&this&new&group&of&organisms?&[2&points]& & & & c) Describe&two&cases&where¤t&shotgun&metagenomics&methods&may¬&detect&an& organism&[4&points]& & &

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