single cell rna sequencing data analysis practical exercises · case, update your gitrepo with...

SinglecellRNAsequencingdataanalysisPracticalexercises

Åsa Björklundasa.bjorklund@scilifelab.se

Practicalonzoom

• Workingroupsof5,theideaisthatyoucanhelpeachotheroutanddiscussresults.

• Onezoombreakoutroompergroup,youshouldhavealreadybeenassignedtoagroupinslack.Pleasestayonlineinthebreakoutroomforyourgroup.

• Cometomainroomforquestionortellusonslackanwecancometoyourbreakoutroom.

• Wehavesomeextraroomswherewecansitwithastudentiftherearespecificquestions.

https://nbisweden.github.io/workshop-scRNAseq/exercises

Threemainpipelinesforanalysing singlecelldata:• Seurat:

• Rbased,centeredaroundSeuratobjects.• Mainlydevelopedfordropletbaseddata• Easytouse,recommendedforRbeginners• Cons:usesaLOTofmemory

• Scran:• Rbased,centeredaroundSingleCellExperiment objects• Hasmoredifferentstatisticalmethods• Canhandlespike-ins• Cons:MorecomplicatedthanSeurattorun.

• Scanpy:• Pythonbased• Handleslargedatasetsbetter.• Cons:Requiresquitesomepythonknowledge.DoesnotyethaveallthefunctionalityoftheRbasedtools

Seuratobject

https://github.com/satijalab/seurat/wiki/Seurat

SingleCellExperiment (SCE)objects

https://bioconductor.org/packages/release/bioc/vignettes/SingleCellExperiment/inst/doc/intro.html

AnnData (Scanpy)objets

https://anndata.readthedocs.io/en/latest/anndata.AnnData.html

Whattochose?

• Itisrecommendedthatyougothroughallthestepswithonepipelineaseachexercisedependsonsavedobjectsfromthepreviousstep.

• Everyoneworksinverydifferentpace.Focusononeofthepipelinesfirst.Ifyouhavetimeleftover,youcanalsotryouttheotherones.

Thedatasets– Covid-19PBMCs

• Elderlypatientsusuallydevelopseverelunginflammationandlungdisfunction.

• Manycelltypesorchestratetheimmuneresponsetothevirus.

• Theirrelativecontributionatthesingle-cellresolutionisstillunclear

GOAL:Whichcelltypesandgenesarealteredwhencomparingbloodimmunecellsfromhealty vsdisease?

Thedatasets– Covid-19PBMCs

• Datafrompaper:”Immunophenotyping ofCOVID-19andinfluenzahighlightstheroleoftypeIinterferonsindevelopmentofsevereCOVID-19”Leeetal.SciImmuno

• Wehaveselected3controlsand3severecovidsamplesandsubsampledto1500cellspersubjectforcomputationalspeed/memory.

• STandtrajectorylabwillbewithotherdatasets.

Installationofallpackages

• Wehavecreatedaconda environmentforthecoursethatshouldcontainallpackagesyouneedfortheexercises

• However,forslingshottrajectoryinferencelab,thereisanadditionalconda environmentthatneedstobeinstalled.

• IfyouchosetoinsteadworkwithstandardRinstallations,youcanusethelistofrequiredpackagesintheenvironmentfileandinstallthemonyourown.

Whyconda?

• OfteneasierinstallationscomparedtotraditionalRinstallationforpackageswithC-compilationetc.

• Goodwaytomanagedifferentversionsofpackagesindifferentprojects.

• Thereareotherwaysofmanagingpackages.E.gpackratforR,pyenv forpythonetc.

Thecode:

• AllcodefortheexercisesisavailableasR-markdowndocuments,orjupyter notebooks,inthefolder:workshop-scRNAseq/labs/compiled/

• Pleasereporttousifyoufindanyerrorsinthecode!– Slackchannel#exercises– AnIssueonthegithub page.

• Wemayfindbugsandupdatethecode– inthatcase,updateyourgit repowithcommand”gitpull”

Reproduciblecoding

• Youshouldalwaysbeabletofindandrecreatetheresults.– Scriptsshouldbeabletorunfrominputfilestocreatetheoutput.

– NeverworkwithsavedRsessions!

• Nameyourscriptswithrelevantnamessoyoucanfindthem2yearslaterJ

• Alwaysbackupcode– goodideatousegithub thatalsogivesyouversioncontrol.

MemoryissuesinR

• scRNAseq datasetsareoftenlarge,thinkabouthowyoucode.Avoidduplicatingobjects!

• Removeunusedmatricesandclearmemorywithgc().

• MostpackagesstorescRNAseq assparsematrices,willrequirepackageMatrixorsimilarformanystandardmatrixoperations.

• IfyoustillhaveissueswithmemoryinR,testsettinge.g.R_MAX_VSIZE=70Gb inthe.Renviron file.Defaultis16Gb.

Troubleshooting

• Slackchannel- #exercises

• Itisimportantthatyoulearnhowtotroubleshootyourselves.– Lookatyourerrormessages,perhapstheansweristhere?– Ifnot– Googleisyourbestfriend!ForumslikeSeqanswers,Stackexchange,Bioconductorsupportforum,specificforums(orgithub issues)foreachpackagemayhavetheanswer.

• TAsaretheretoansweranyquestionsandgivesuggestions,butwemaynotalwayshavetheanswer.

https://nbisweden.github.io/single-cell-pbl/glossary_of_terms_single_cell.html

Rmarkdown demonstration