A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor

Aaron T L LunJohn C Marioni


Single-cell RNA sequencing (scRNA-seq) is widely used to profile the transcriptome of individual cells. This provides biological resolution that cannot be matched by bulk RNA sequencing, at the cost of increased technical noise and data complexity. The differences between scRNA-seq and bulk RNA-seq data mean that the analysis of the former cannot be performed by recycling bioinformatics pipelines for the latter. Rather, dedicated single-cell methods are required at various steps to exploit the cellular resolution while accounting for technical noise. This article describes a computational workflow for low-level analyses of scRNA-seq data, based primarily on software packages from the open-source Bioconductor project. It covers basic steps including quality control, data exploration and normalization, as well as more complex procedures such as cell cycle phase assignment, identification of highly variable and correlated genes, clustering into subpopulations and marker gene detection. Analyses were demonstrated on gene-level count data from several publicly available datasets involving haematopoietic stem cells, brain-derived cells, T-helper cells and mouse embryonic stem cells. This will provide a range of usage scenarios from w...Continue Reading


Mar 3, 2006·PLoS Biology·Hilary A CollerJames M Roberts
Nov 21, 2007·Bioinformatics·Peter LangfelderSteve Horvath
Mar 4, 2010·Genome Biology·Mark D Robinson, Alicia Oshlack
May 13, 2010·Proceedings of the National Academy of Sciences of the United States of America·Richard BourgonWolfgang Huber
Oct 29, 2010·Genome Biology·Simon Anders, Wolfgang Huber
Nov 4, 2010·Statistical Applications in Genetics and Molecular Biology·Belinda Phipson, Aaron T L Lun
Apr 6, 2013·Nucleic Acids Research·Yang LiaoWei Shi
Jul 24, 2013·Nature Reviews. Molecular Cell Biology·Cosetta BertoliRobertus A M de Bruin
Sep 24, 2013·Nature Methods·Philip BrenneckeMarcus G Heisler
Dec 24, 2013·Nature Methods·Saiful IslamSten Linnarsson
Jan 5, 2014·Nature Protocols·Simone PicelliRickard Sandberg
Feb 4, 2014·Genome Biology·Charity W LawAaron T L Lun
Dec 18, 2014·Genome Biology·Michael I LoveSimon Anders
Jan 22, 2015·Nucleic Acids Research·M E RitchieAaron T L Lun
Jan 30, 2015·Nature Reviews. Genetics·Oliver StegleJohn C Marioni
Jan 31, 2015·Nature Methods·Wolfgang HuberMartin Morgan
Jun 25, 2015·PLoS Computational Biology·Catalina A VallejosSylvia Richardson
Jul 6, 2015·Methods : a Companion to Methods in Enzymology·Antonio ScialdoneFlorian Buettner
Aug 25, 2015·Nature Methods·Ning LengChristina Kendziorski
Oct 3, 2015·Cell Stem Cell·Aleksandra A KolodziejczykSarah Amalia Teichmann
Dec 17, 2015·Bioinformatics·Philipp AngererFlorian Buettner
Dec 18, 2015·F1000Research·Michael I LoveWolfgang Huber
Feb 19, 2016·Genome Biology·Tomislav IlicicSarah Amalia Teichmann
Apr 5, 2016·Nature Biotechnology·Nicolas L BrayLior Pachter
May 15, 2016·Nucleic Acids Research·Zhicheng Ji, Hongkai Ji


Feb 19, 2019·Pediatric Research·Qian WangThomas J Mariani
Feb 27, 2018·Genome Biology·Koen Van den BergeLieven Clement
Feb 23, 2019·Nature·Blanca Pijuan-SalaBertie Göttgens
Mar 11, 2018·BMC Bioinformatics·Jesse M ZhangDavid N Tse
Jan 9, 2019·Nature Reviews. Genetics·Vladimir Yu KiselevMartin Hemberg
Aug 8, 2017·Nature Immunology·Michael DelacherMarkus Feuerer
Apr 6, 2019·Database : the Journal of Biological Databases and Curation·Oscar FranzénJohan L M Björkegren
Jul 31, 2018·The FEBS Journal·Helena Todorov, Yvan Saeys
May 23, 2018·Nature Genetics·Nathan G SkeneJens Hjerling-Leffler
Dec 1, 2017·Nature Protocols·Cathryn R CadwellAndreas Savas Tolias
Nov 2, 2017·Proceedings of the National Academy of Sciences of the United States of America·Lihe ChenMark A Knepper
Jun 22, 2019·Nucleic Acids Research·Jurrian K de KanterFrank C P Holstege
Jun 27, 2018·Nature Methods·Mo HuangNancy R Zhang
Sep 4, 2019·Nature Communications·Sung Pil HongLuca Magnani
Jul 12, 2018·Nature Communications·Jonathan A GriffithsJohn C Marioni
Feb 27, 2018·Briefings in Bioinformatics·Shun H YipJunwen Wang
Jan 26, 2018·Nature Communications·Stéphanie KilensMilieu Intérieur Consortium
Aug 11, 2018·PLoS Computational Biology·Luyi TianM E Ritchie
Jan 22, 2019·Nature Communications·Tsukasa KounoJay W Shin
Jul 18, 2018·Nature Immunology·Arianne C RichardGillian M Griffiths
Dec 13, 2019·Nature·Ricard ArgelaguetWolf Reik
Dec 23, 2018·Nature Methods·Maren BüttnerFabian J Theis
Dec 23, 2018·Nature Communications·Miguel GanuzaShannon McKinney-Freeman
Dec 8, 2019·Cells·Ahmed GhallabJan G Hengstler
Jan 25, 2019·Genome Medicine·Alexander N CombesMelissa H Little
Nov 24, 2019·BMC Bioinformatics·Ilaria PatuzziBarbara Di Camillo
Jan 27, 2019·PloS One·Shannon R McCurdyLior Pachter
Feb 8, 2018·Genome Biology·F Alexander WolfFabian J Theis
Apr 24, 2020·Bioinformatics·Oscar Franzén, Johan L M Björkegren
Mar 17, 2020·Bioinformatics·Emanuele AlivertiDavid B Dunson
Sep 9, 2019·GigaScience·Luca AlessandrìRaffaele A Calogero
Jul 7, 2019·Nature Communications·Daphne TsoucasGuo-Cheng Yuan
Dec 25, 2019·Basic Research in Cardiology·Payam Haftbaradaran EsfahaniRalph Knöll
Jul 14, 2020·Bioinformatics·Kerem Wainer-Katsir, Michal Linial
Jun 10, 2020·Toxicological Sciences : an Official Journal of the Society of Toxicology·Kevin S HsuRichard I Enelow
Aug 1, 2020·The Journal of Experimental Medicine·Kirby D JohnsonEmery H Bresnick
Aug 23, 2019·Nature Communications·Zhengtao XiaoJason W Locasale
Aug 26, 2020·ELife·Jeanette Baran-GaleGeorg A Holländer
Feb 16, 2019·The Journal of Experimental Medicine·Fabian KleinPanagiotis Tsapogas
Jun 11, 2019·PeerJ·Khadija El AmraniMiguel A Andrade-Navarro
Dec 25, 2019·Genome Biology·F William TownesRafael A Irizarry
Jan 21, 2020·Genome Biology·Koki TsuyuzakiItoshi Nikaido
Jan 25, 2020·Science·Gunsagar S GulatiAaron M Newman
Apr 23, 2020·Frontiers in Oncology·Guillermo de Anda-Jáuregui, Enrique Hernández-Lemus
Mar 15, 2020·Communications Biology·Jackson A HoffmanTrevor K Archer
Aug 29, 2020·Genome Biology·Wenpin HouStephanie C Hicks
May 12, 2018·Diagnostics·Francesc Castro-GinerNicola Aceto
Dec 14, 2018·Frontiers in Cardiovascular Medicine·Andrea MassaiaMichela Noseda
Jun 4, 2019·Frontiers in Immunology·Beth A Jiron TamburiniMatthew A Burchill
Jan 4, 2020·Scientific Data·Jinling LiaoZengnan Mo
Nov 18, 2018·Frontiers in Immunology·Wendi A BaconD Stephen Charnock-Jones
Dec 6, 2019·Frontiers in Cellular and Infection Microbiology·Regan J HaywardGarry S A Myers
Apr 9, 2020·Frontiers in Cellular Neuroscience·Jinting GuanGuoli Ji
Sep 1, 2019·Cells·Sne Morag, Mali Salmon-Divon
Aug 6, 2020·BMC Medical Genomics·Stefanie Friedrich, Erik L L Sonnhammer
Apr 25, 2019·F1000Research·Brendan T Innes, Gary D Bader
Jan 10, 2021·Journal of Cell Science·Krzysztof KolmusMarta Miączyńska
Nov 20, 2020·Computational and Structural Biotechnology Journal·Yaru ZhangJianzhong Su
Jan 8, 2021·Journal of Translational Medicine·Jinting GuanGuoli Ji
Jan 9, 2021·Science·Richard C V TyserShankar Srinivas

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Cell Cycle
Complete Blood Count
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