• The best place to start is to learn more about MSK-ACCESS and for that please read the paper:

  • Brannon, A. R. et al. Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell- free DNA via paired normal sequencing using MSK-ACCESS. Nat Commun 12, 3770 (2021).

• Learn more about the current collapsing method Marianas

• Learn more about the new collapsing (Nucleo) method using Fgbio

• Learn more about the Quality Control V1

• Understand the updated version for the above using these Quality Control V2

• Learn about ACCESS Data analysis scripts that help with downstream analysis

• Learn about IGV for viewing BAM files to distinguish real variants from artifacts

Project Management

Functional Resources

Path's to know

CMO-ACCESS

Data on JUNO for CMO-ACCESS samples

Resources

Details of CMO ACCESS Resources

• admie - Files used for microsatellite instability detection tool ADMIE for MSK-ACCESS

• cosmic - VCF file of cosmic used in MSK-ACCESS workflows

• dbSNP - VCF file of dbSNP used in MSK-ACCESS workflows

• exac - VCF file of ExAC used in MSK-ACCESS workflows

• mills-and-1000g - VCF file of mills-and-1000g used in MSK-ACCESS

• reference - reference genome file used in MSK-ACCESS workflows

• tools - general packages used in MSK-ACCESS workflows

• msk-access - Data-specific resources for MSK-ACCESS workflows. This includes the following:

  • hiseq4000_curated_duplex_bams_dmp - curated DMP duplex BAMS from HiSeq 4000

  • novaseq_curated_simplex_bams_dmp - curated DMP simplex BAM from NovaSeq.

  • hiseq4000_curated_simplex_bams_dmp - curated DMP simplex BAM from HiSeq 4000

  • novaseq_curated_standard_bams_dmp - curated DMP standard BAM from NovaSeq

  • hiseq4000_curated_standard_bams_dmp - curated DMP standard BAM from HiSeq 4000

  • novaseq_curated_unfiltered_bams_dmp - curated DMP unfiltered BAM from NovaSeq

  • hiseq4000_curated_unfiltered_bams_dmp - curated DMP unfiltered BAM from HiSeq 4000

  • novaseq_unmatched_normal_plasma_duplex_bams_dmp - DMP unmatched normal plasma duplex BAM from NovaSeq

  • hiseq4000_unmatched_normal_plasma_duplex_bams_dmp - DMP unmatched normal plasma duplex BAM from HiSeq 4000

  • novaseq_unmatched_normal_plasma_standard_bams_dmp - DMP unmatched normal plasma standard BAM from NovaSeq

  • hiseq4000_unmatched_normal_plasma_standard_bams_dmp - DMP unmatched normal plasma standard BAM from HiSeq 4000

  • novaseq_curated_duplex_bams_dmp - curated DMP duplex BAMS from NovaSeq

  • regions_of_interest - Different interval files describing regions of interest for MSK-ACCESS assay

What is CMO-CH assay?

CMO-CH is offered by the Center for Molecular Oncology (CMO) to MSK researchers for profiling white blood cell DNA to detect mutations in the most commonly altered clonal hematopoiesis (CH) associated genes.

What is the panel design?

• 596 targets capturing 58% of CH and 90.4% of CH-PD mutations identified in the latest CH dataset from 40K patients

• Total size = 1,143 probes (0.14 Mb)

• Full gene coverage for TP53, TET2, ASXL1, DNMT3A, PPM1D, CHEK2, ASXL1, ATM, SF3B1, SRSF2, U2AF1, and U2AF2•Additional targets with hotspot positions from IMPACT heme assay

• SNP tiling around TP53, CBL, MPL, JAK2, EZH2, TET2, RUNX1, and ATM (+/-10kb) to identify allelic-imbalances

• 40 fingerprint SNPs that are shared with all other NGS assays (IMPACT, ACCESS, WES etc.) to detect sample mismatches

This pages

What is MSK-ACCESS?

It is a hybrid capture panel designed for Analysis of Circulating cfDNA to Evaluate Somatic Status using the Unique Molecular Index (UMIs) for high sensitivity. MSK-ACCESS is 13% as large, captures 47% of all mutations detected by MSK-IMPACT.

Brannon, A. R. et al. Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell- free DNA via paired normal sequencing using MSK-ACCESS. Nat Commun 12, 3770 (2021).

What is the panel design?

Selected exons of 129 genes for mutation detection

• OncoKB Level 1-4

• Hotspot sites

• High rates of mutations

• Protein kinase domains

• Tumor suppressor genes

Microsatellite regions

SNPs of zygosity & copy number of 12 genes

Common SNPs for genome-wide copy number

Introns for structural variants of 10 genes

Clonal hematopoiesis genes

What are the Design Implications?

• Matched cfDNA-WBC (”tumor-normal”) assay to detect somatic alterations

• Sensitivity for mutation calling depends on ‘duplex’ collapsed coverage

• Different sensitivities for different classes of alterations

  • Genotyping +++++

  • De novo mutations, indels ++++

  • MSI +++

  • Rearrangements +++

  • Copy number ++

  • Tumor mutation burden ○

BAM Generation & Quality Control

Github Location -> https://github.com/mskcc/ACCESS-Pipeline/blob/master/workflows/ACCESS_pipeline.cwl

Tools Used:

• BWA

• Trimgalore

• GATK

• Picard Tools

• ABRA2

• Marianas

Variant Calling

Small Variants

Github Location -> https://github.com/mskcc/ACCESS-Pipeline/blob/master/workflows/subworkflows/snps_and_indels.cwl

Tools Used:

• VardictJava

• MuTect

• VCF2MAF

Copy Number Variant (CNV)

Github Location -> https://github.com/mskcc/ACCESS-Pipeline/blob/master/workflows/subworkflows/call_cnv.cwl

Refer to Bioinformatics Pipeline to Detect CNA's section in this paper for details:

Dara S. Ross, Ahmet Zehir, Donavan T. Cheng, Ryma Benayed, Khedoudja Nafa, Jaclyn F. Hechtman, Yelena Y. Janjigian, Britta Weigelt, Pedram Razavi, David M. Hyman, José Baselga, Michael F. Berger, Marc Ladanyi, Maria E. Arcila, Next-Generation Assessment of Human Epidermal Growth Factor Receptor 2 (ERBB2) Amplification Status: Clinical Validation in the Context of a Hybrid Capture-Based, Comprehensive Solid Tumor Genomic Profiling Assay, The Journal of Molecular Diagnostics, Volume 19, Issue 2, 2017, Pages 244-254, ISSN 1525-1578, https://doi.org/10.1016/j.jmoldx.2016.09.010.

Structural Variant (SV)

Github Location -> https://github.com/mskcc/ACCESS-Pipeline/blob/master/workflows/subworkflows/manta.cwl

Tool Used:

• Manta

• iAnnotateSV

Microsatellite Instability Status (MSI)

Github Location -> https://github.com/mskcc/ACCESS-Pipeline/blob/master/workflows/subworkflows/msi.cwl

Tool Used:

• ADMIE

Configurations

Voyager has all our configurations in the jinja template, it includes all the paths for various files and tools associated with the workflows, all location are on JUNO:

snps_indels: beagle/input_template.json.jinja2 at master · mskcc/beagle (github.com)

CNV: beagle/input_template.json.jinja2 at master · mskcc/beagle (github.com)

Fastq_to_bam: beagle/input_template.json.jinja2 at master · mskcc/beagle (github.com)

MSI: beagle/input_template.json.jinja2 at master · mskcc/beagle (github.com)

SV : beagle/input_template.json.jinja2 at master · mskcc/beagle (github.com)