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CNA workflow on IR for amplifications

Author: Giorgia Mori

Rationale

Preliminary analyses by Giorgia Mori suggested that the Ion Reporter pipeline described below performs well to call amplifications. No IR pipelines were found to call deletions on the samples used for benchmarking (mainly Horizon samples). For deletions it was found that CNVkit may perform better (see this presentation). Therefore, it was proposed to use the Ion reporter CNA pipeline described below to call amplifications and CNVkit to call deletions.

The Target Region File (.bed file)

For CNA call, the Target Region File (bed file) should contain this mandatory field:

GENE_ID=name

which should be the 5th column. Here you have to specify the name of the gene present in the amplicon.

PURPUSE=CNV

is optional and should be the 6th column. It works like a ‘hotspot’ for CNA call.

Your .bed file should look like this:


chr1	40362511	40362630	MYCL1_13.3.70	GENE_ID=MYCL;Pool=1	PURPOSE=CNV

The ACC lung Target Regions File used in the workflow wfCNA-06-IR5.10 and modified for CNA call is named WG_IAD97207.20161004.spikeinCL.designedTRIS and can be downloaded from the link present in the table below.

The Baseline

The baseline CNVBase_18_male_samples used in wfCNA-08-IR5.10 was trained using 18 male samples. You can download this baseline from the link present in the table below.

If you want to create your own baseline see - Appendix -> Create a CNA baseline.

The Filter Chain

For somatic CNA call, a custom Filter Chain should be set before starting with workflow creation. On IR, click on:

Workflows -> Presets -> Create Preset -> Filter Chain

Give a name to the Filter Chain (e.g MyFilterChain)
Select hg19 Reference
Select CNV Confidence Range - CNVs Only
Do not modify default parameters and click on ‘Set’
Click Save

Screen_Shot_2018-09-03_at_15.58.26

Now you can start creating your workflow.

N.B. in order to reduce False Positives (FPs) call you can modifiy the range of the Filter Chain choosing as the lower value 30 instead of 10. If you have a sample with a subclonal mutation, choose as lower value 20 instead of 10.

Workflow description

This is the description of the wfCNA-08-IR5.10 workflow used to call CNA. Click on the links to download the files exported from IR 5.10.


WorkflowID	wfCNA-08-IR5.10
Workflow description	Single sample CNA workflow
Research application	DNA
Sample groups	Single
Reference genome	hg19
Reference panel design	ACC Lung target region file WG_IAD97207.20161004.spikeinCL.designedTRIS
Annotation	all
MyVariant database	MyVariantDefault_hg19
Filter chains	MyFilterChain - CNV Confidence Range - CNVs Only (10.0 <= CNV Confidence Range - CNVs Only <= 1.0E7)
Baseline	CNVBase_18_male_samples
Final Report	default
Parameters	default
IR version	5.10
IR account username	Todaro.Vincenzo@20mail.it
Created by	Giorgia Mori
Creation date	06/07/2018
Other comments	Validation results: All amplifications detected. No deletions detected.