<section data-background-image="https://maxulysse.github.io/assets/img/background/Sarek-Park-01.jpg" data-background-opacity=0.5 > # Sarek and rnafusion ## Detect variants and fusion genes Maxime Garcia <small> [<i class="fab fa-twitter"></i> @gau](https://twitter.com/gau) [<i class="fab fa-github"></i> @MaxUlysse](https://github.com/MaxUlysse) [<i class="fa fa-globe-europe"></i> maxulysse.github.io](https://maxulysse.github.io/) </small> DNA Club <small> Science for Life Laboratory, Stockholm - 2020/09/16 </small> --- [](https://ki.se/forskning/barntumorbanken) <!-- .element class="image-H10" --> The Swedish Childhood Tumor Biobank (BTB) [](https://ki.se) <!-- .element class="image-H10" --> Note: * Working for The Swedish Childhood Tumor Biobank located at KI --- [](https://scilifelab.se/) <!-- .element class="image-H10" --> National centre for molecular biosciences with focus on health and environmental research [](https://ki.se/) <!-- .element class="image-H75" --> | [](https://www.kth.se/) <!-- .element class="image-H75" --> | [](https://www.su.se/) <!-- .element class="image-H75" --> | [](https://www.uu.se/) <!-- .element class="image-H75" --> :-:|:-:|:-:|:-: Note: * SciLifeLab is several infrastructures === [](https://ngisweden.scilifelab.se/) <!-- .element class="image-H10" --> * State-of-the-art infrastructure * Sequencing (DNA, RNA ...) * Guidelines and support * Sample collection, study design, protocol selection * Bioinformatics analysis Note: * NGI is a sequencing facility used by researchers all over Sweden === [](https://www.nbis.se/) <!-- .element class="image-H10" --> * Swedish ELIXIR node * Bioinformatics support for Swedish researchers --- ## Reproducibility is central [](https://academic.oup.com/view-large/figure/118918033/giy077fig1.jpg) <!-- .element class="image-50" --> [<i class="ai ai-doi"></i> 10.1093/gigascience/giy077](https://doi.org/10.1093/gigascience/giy077) Note: * For me, as a bioinformatician it is a crucial matter --- ## What is Sarek <section data-background-image="https://maxulysse.github.io/assets/img/background/Sarek-beer.jpg" data-background-opacity=0.5 > <div class="fragment fade-in" data-fragment-index="2"> A [National Park](https://www.sverigesnationalparker.se/en/choose-park---list/sarek-national-park/) in Northern Sweden. <small> * Long, deep, narrow valleys and wild, turbulent water * A tortuous delta landscape * Completely lacking in comfortable accommodations * One of Sweden’s most inaccessible national parks * There are no roads leading up to the national park </small> </div> --- <section data-background-image="https://maxulysse.github.io/assets/img/background/Sarek-Park-02.jpg"> --- <section data-background-image="https://maxulysse.github.io/assets/img/background/Sarek-Park-02.jpg" data-background-opacity=0.5 > * [Ruotes](https://github.com/SciLifeLab/Sarek/releases/tag/2.1.0) * [Skårki](https://github.com/SciLifeLab/Sarek/releases/tag/2.2.0) * [Äpar](https://github.com/SciLifeLab/Sarek/releases/tag/2.3) * [Ålkatj](https://github.com/nf-core/sarek/releases/tag/2.5) * [Årjep-Ålkatjjekna](https://github.com/nf-core/sarek/releases/tag/2.5.1) * [Jåkkåtjkaskajekna](https://github.com/nf-core/sarek/releases/tag/2.5.2) * [Piellorieppe](https://github.com/nf-core/sarek/releases/tag/2.6) * [Gådokgaskatjåhkkå](https://github.com/nf-core/sarek/releases/tag/2.6.1) --- [](https://nf-co.re/sarek) <!-- .element class="image-H10" --> <div class="fragment fade-in" data-fragment-index="2"> * Open-Source Nextflow Pipeline * Started at NGI * In collaboration with NBIS * Support from BTB * In collaboration with QBiC (Tübingen) </div> === [](https://www.nextflow.io/) <!-- .element class="image-50" --> * Workflow manager * Data driven language * Portable * executable on multiple platforms * Shareable and reproducible * with containers or virtual environments Note: * Early adopters of Nextflow for its portability, shareability and of course reproducibility --- ## Multiple flavors  <!-- .element class="image-10" --> <div class="fragment fade-in" data-fragment-index="2">  <!-- .element class="image-10" --> |  <!-- .element class="image-10" --> :-:|:-: </div> === ## WES and Targeted Sequencing  <!-- .element class="image-25" --> === ## Reference genomes [AWS iGenomes](https://registry.opendata.aws/aws-igenomes/) * GRCh37 * GRCh38 * GRCm38 --- ## Preprocessing [](https://software.broadinstitute.org/gatk/best-practices/) <!-- .element class="image-H25" --> Based on GATK Best Practices (GATK 4.1.7.0) <div class="fragment fade-in" data-fragment-index="2"> * Reads mapped to reference genome with `bwa` * Duplicates marked with `picard MarkDuplicates` * Recalibrate with `GATK BaseRecalibrator` </div> === ## Germline Variant Calling * SNVs and small indels * HaplotypeCaller * Strelka2 * Freebayes * mpileup * Structural variants * Manta * TIDDIT === ## Somatic Variant Calling * SNVs and small indels * Mutect2 * Strelka2 * Freebayes * Structural variants * Manta * Sample heterogeneity, ploidy and CNVs * ASCAT * Control-FREEC * Microsatellite instability * MSIsensor === ## Annotation * VEP and SnpEff * <i class="fas fa-database"></i> ClinVar, COSMIC, dbSNP, GENCODE, gnomAD, polyphen, sift, etc. === ## Reports [](https://multiqc.info/) <!-- .element class="image-H25" --> --- ## Workflow [](https://github.com/nf-core/sarek/releases/tag/2.6.1) <!-- .element class="image-25" --> --- ## Prioritization * First step towards clinical use * Rank scores are computed for all variants * COSMIC, ClinVar, SweFreq and MSK-IMPACT (cancerhotspots.org) * Findings are ranked * Well known, high-impact variants * Variants in known cancer-related genes * Remaining variants --- ## What is coming soon * <i class="far fa-check-square"></i> `@sarek-team` to mention the core sarek developers on Slack * <i class="far fa-check-square"></i> `@nf-core/sarek` to mention the core sarek developers on GitHub * <i class="far fa-check-square"></i> BWA-MEM2 ([dev](https://github.com/nf-core/sarek/tree/dev)) * <i class="far fa-check-square"></i> Bug-fixes ([dev](https://github.com/nf-core/sarek/tree/dev)) * <i class="far fa-square"></i> DSL 2 ([dsl2](https://github.com/nf-core/sarek/tree/dsl2)) with [@ggabernet](https://github.com/ggabernet) and [@FriederikeHanssen](https://github.com/FriederikeHanssen) === ## What is coming next * Validation tests * More tools * Sub-workflows for specific usage * Improved cloud usage * Improved usage for non-model organism * Joint Variant Calling * More downstream processing of the final vcf files * Easier connection to [Scout](https://www.clinicalgenomics.se/scout/) Note: * Scout is a tool developed by SciLifeLab Clinical Genomics to analyse VCF files --- ## Sarek usage * Within BTB * Tumor/normal pairs * In production at NGI * All normal samples * Tumor/normal pairs * The whole SweGen dataset * 1 000 normal samples (GRCh38) * Genome Medicine Sweden Note: * GMS is an initiative to implement Precision Medicine at a national level --- ## Publication in F1000Research Sarek: A portable workflow for whole-genome sequencing</br> analysis of germline and somatic variants</br> [version 2; peer review: 2 approved] <small> Maxime Garcia, Szilveszter Juhos, Malin Larsson, Pall I. Olason, Marcel Martin,</br> Jesper Eisfeldt, Sebastian DiLorenzo, Johanna Sandgren, Teresita Díaz De Ståhl,</br> Philip Ewels, Valtteri Wirta, Monica Nistér, Max Käller, Björn Nystedt </small> [<i class="ai ai-doi"></i> doi.org/10.12688/f1000research.16665.2](https://doi.org/10.12688/f1000research.16665.2) --- [](https://nf-co.re/rnafusion) <!-- .element class="image-H10" --> <div class="fragment fade-in" data-fragment-index="2"> * Open-Source Nextflow Pipeline * Part of nf-core * Started at NGI * Support from BTB </div> <div class="fragment fade-in" data-fragment-index="3"> <small> Project initiated by [Rickard Hammarén](https://github.com/Hammarn), fully matured under [Martin Proks](https://github.com/matq007), shamelessly taken over by me. </small> </div> === Run and gather outputs from * Arriba * EricScript * FusionCatcher * Pizzly * Squid * STAR-Fusion <div class="fragment fade-in" data-fragment-index="2"> Generate a nice final report </div> --- ## What is coming next in rnafusion * Validation tests * More tools * DSL2 * More genomes * Open to non-cancer specific usage --- ## Get involved * Our code is hosted on Github * [<i class="fab fa-github"></i> github.com/nf-core](https://github.com/nf-core) * [<i class="fab fa-github"></i> github.com/nf-core/sarek](https://github.com/nf-core/sarek) * [<i class="fab fa-github"></i> github.com/nf-core/rnafusion](https://github.com/nf-core/rnafusion) * We have Slack * [<i class="fab fa-slack"></i> nfcore.slack.com](https://nfcore.slack.com/) * [<i class="fab fa-slack"></i> nfcore.slack.com/channels/sarek](https://nfcore.slack.com/channels/sarek) * [<i class="fab fa-slack"></i> nfcore.slack.com/channels/rnafusion](https://nfcore.slack.com/channels/rnafusion) --- <div class="r-stack"> <img src="/assets/img/svg/acknowledgments_2020.svg" title="Acknowledgements" alt="Acknowledgements" class="image-75 fragment fade-out" data-fragment-index="0"/> <img src="/assets/img/svg/institutes_2020.svg" title="Acknowledgements" alt="Acknowledgements" class="image-75 fragment fade-in-then-out" data-fragment-index="0"/> <img src="/assets/img/slides/nf-core_contributors_jobim.png" title="Acknowledgements" alt="Acknowledgements" class="image-75 fragment fade-in-then-out" data-fragment-index="1"/> <img src="/assets/img/slides/nf-core_sarek_contributors_2.6.1.png" title="Acknowledgements" alt="Acknowledgements" class="image-75 fragment fade-in-then-out" data-fragment-index="2"/> <img src="/assets/img/slides/nf-core_rnafusion_contributors_1.2.0.png" title="Acknowledgements" alt="Acknowledgements" class="image-75 fragment fade-in-then-out" data-fragment-index="3"/> </div> --- <section data-background-image="https://maxulysse.github.io/assets/img/background/Stockholm-by-night.jpg" data-background-opacity=0.5 > ## Any questions * [<i class="fa fa-globe-europe"></i> nf-co.re](https://nf-co.re/) * [<i class="fab fa-github"></i> nf-co.re/sarek](https://nf-co.re/sarek) * [<i class="fab fa-slack"></i> #sarek](https://nfcore.slack.com/channels/sarek) * [<i class="fab fa-github"></i> nf-co.re/rnafusion](https://nf-co.re/rnafusion) * [<i class="fab fa-slack"></i> #rnafusion](https://nfcore.slack.com/channels/rnafusion)