In this new release we have incorporated 48 new cohorts to the analysis (Figure 1) representing 4943 new samples (Table 1 and Table 2).

Some previously included cohorts have increased the number of samples they contribute to the analysis. This is the case of cohorts sequenced by the Hartwig Medical Foundation (release downloaded on 21/10/2021) and ICGC (latest release from 2019 downloaded). After careful examination, we have excluded an ICGC cohort (LINC_JP_WGS_ICGC) which was completely included within the HC_PCAWG cohort (now re-named PCAWG_WGS_LIVER_HCC) (Figure 1).

New cohorts have been contributed by some already included data sources, such as TARGET, from which we obtained 2 new cohorts with Neuroblastoma (NB) and Osteosarcoma (OS) samples.

We also included datasets from new genomic data sources, such as CPTAC and CGCI.

Due to changes in the sample annotation of the TCGA_WXS_AML cohort, 95 more samples of this cohort were processed by the pipeline.

In order to standardize the cohort nomenclatures, cohort names have been updated, applying -in most cases- the following pattern:

{PROJECT}_{PLATFORM}_{ONCOTREE_CODE}_{OTHER_INFO}_{YEAR}

Examples: CPTAC_WXS_BRCA_2020, ICGC_WXS_AML_LAML_CN_2019, HARTWIG_WGS_NSCLC_2020

Several updates have been introduced in the IntOGen pipeline:

1. bgparsers replaced with OpenVariant:

We have developed OpenVariant, a new python package to parse the input files. This new method annotates all SNVs and indels from the mutation data files and transforms the data in a standardized format, by reading a yaml file prepared by the user.

Importantly, the previous bgparsers tool had a bug in the annotation of indels, leading to some indels not being processed. This bug has been corrected in OpenVariant. As a consequence, there are, overall, more mutations annotated per sample.

2. Seed to run OncodriveFML, OncodriveCLUSTL, smRegions and dNdScv added:

We have added the use of a seed in these methods to reduce the variability in the calculation of the p-value of genes across IntOGen runs. Due to difficulties in the implementation, we could not add it in the remaining three methods run by the pipeline.

3. Ensembl VEP updated from v92 to v101:

We updated the Ensembl VEP version to 101. This version may contain important gene annotation differences, and therefore the number of mutations annotated may change substantially from the previous release.

4. MSKCC oncotree updated to version 2021:

We have updated the oncotree from the latest version released by MSKCC (November 2021). As a result, the number of cancer type nodes has increased from 82 to 889.

We have applied some modifications to the MSKCC Oncotree:

• NON_SOLID node added after TISSUE and before MYELOID and LYMPHOID
• SOLID node added after TISSUE and before the rest of tissues
• ALL node added after LNM and before TLL and BLL
5. CGC genes updated to v95:

We have updated the Cancer Gene Census gene list with the v95. This version distinguishes between Tier1 and Tier2 CGC genes. In the IntOGen pipeline, both tiers are considered as a unique list of CGC genes.

6. Hugo Symbols updated to the 20/01/2022 release:

We have unified all gene SYMBOLs according to the latest version of the HUGO Gene Nomenclature Committee.

7. CancerMine database updated to 07/12/2021 release:

The CancerMine database, which gathers information on literature evidence for cancer genes, was updated.

8. CADD update:

CADD, a tool for scoring the deleteriousness of single nucleotide variants - as well as insertion/deletions variants in the human genome, was updated from version 1.4 to version 1.6.

9. List of frequent artifacts updated:

In the previous release the pipeline was using a list of ‘Known Artifacts’ and a separated blacklist of genes, to rule out possible false positives in the post-processing. In this release, the blacklist genes are considered ‘Known Artifacts’, and, together with the ‘Warning artifacts’ (list of genes suspected to be artifacts) are all excluded in the post-processing step. Moreover, the olfactory receptors genes list from HORDE database used as a filter was updated to to Build #44c (30/July 2019).

10. File with unfiltered drivers included:

In this new release we include a list of unfiltered drivers as one of the outputs of the IntOGen pipeline. This file annotates all the filters applied to the post-processing step: from the output of the combination to the final set of driver genes. [unfiltered_drivers.tsv]

11. BoostDM connection prepared:

We have included along with the IntOGen output, all the files needed to run BoostDM. Specifically, two steps in the pipeline were added:

• DriversSaturation: all possible mutations for a given gene are generated and returned as independent annotated files per gene. [{gene}.vep.gz]
• filterMNVs: one file containing all positions detected as MNVs. [mnvs.tsv.gz]

In the new release, several aspects have been improved: there are more cohorts and tumor type in which a gene is identified as driver; and more drivers and cohorts per tumor type (Figure 2).

Cohorts per driver Cohorts per cancer type Drivers per cancer type Cancer types per driver

The new release contains a total of 618 driver genes, 50 more than the previous release. There are 7 new tumor types: two of them are from two cohorts with re-assigned tumor type (from PAAD to PANCREAS on HARTWIG_PANCREAS and from RCCC to RCC on HARTWIG_KIDNEY_RENAL_CELL) and five of them are new; PSCC (Penil Squamous Cell Carcinoma, HARTWIG_WGS_PSCC_2020), MCC (Merkel Cell Carcinoma, HARTWIG_WGS_MCC_2020), COAD (Colon Adenocarcinoma, CPTAC_WXS_COAD_2020) and MLYM (Malignant Lymphoma, ICGC_WGS_MLYM_MALY_DE_PED_2019)

Among these 618 driver genes, 133 that are new, while 82 included in the previous release are no longer identified as drivers (Figure 3). This is due to several reasons:

• Different mutation annotations:
• Increase in the number of processed indels (see above)
• New Ensembl gene annotation, where annotation of mutations per gene may have changed with respect to the previous release
• Different bona fide cancer gene / artifacts lists:
• CGC genes: The step of combination in the pipeline depends on the list of bona fide cancer genes obtained from CGC.
• CancerMine database: This database, that annotates literature-gathered information, is used to provide evidence of the involvement of genes in tumorigenesis.
• Lists of artifacts: in this release we filter out in the post-processing step all known artifacts and suspect/warning artifacts. The previous release was just filtering the known artifacts. These are accordingly annotated in the unfiltered drivers file.
• The final ranking q-value list:
• Implementation of a seed: Everytime we run Intogen, the final ranking of q-values may change, due to heuristic calculation of the background model in different methods. This means that genes that are usually found close to the driver threshold may have driver or passenger status across runs. In this release, we included a seed option for 4 methods (those that allowed it), to reduce the variability across runs. This problem is still not solved, as there are still 3 methods where the background calculation cannot be fixed with a seed.

We provide a table summarizing the reason why genes previously identified as drivers do not appear in the current release (See here). For further inquiries on the reason why a driver is not appearing in this release, please send a request to (bbglab@irbbarcelona.org) and we will try to provide a more detailed explanation.