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Set Analysis Parameters

Open DNA Amplicon from BaseSpace™ Sequence Hub as follows.

Select the Apps tab, and then select DNA Amplicon.

From the Version drop-down list, select 2.0.0.

Select Launch Application.

To override the default analysis name, enter a preferred analysis name in the Analysis Name field.

The default is the app name with the date and time the session started.

From the Save Results To field, select Select Project, and then select a project to store app results to.

From the Biosample(s) field, select the biosample you want to analyze as follows.

Select Select Biosample(s).

Select the checkbox of each biosample you want to analyze, and then select Select.

The labels of this field and button in the software interface might be different from the labels in this guide, depending on the BaseSpace™ Sequence Hub mode. In New mode, all data that is associated with the selected biosample is used in the analysis.

[Optional] From the Custom Genome (Optional) field, select Select Dataset File(s), and then browse to the FASTA file you want to use as the custom genome.

From the Targeted Amplicons drop-down list, select a panel of targeted amplicons representative of the selected samples.

If you selected Custom Panels from Targeted Amplicons, select Select Dataset File(s), and then select a manifest file. If the manifest file you want to use is not listed, import the file as follows.

Open a new browser window and visit the BaseSpace™ Sequence Hub home page.

Select Projects from the My Data tab.

Select the project you want to upload the manifest file to.

Select File | Upload | Files | Manifest.

Follow the onscreen instructions to add the Custom Amplicon manifest file (*.txt) to the project. Make sure that the reference genome is specified in the header of the manifest file. See Reference Genomes on page 1.

Return to the DNA Amplicon app and repeat step 7 to select the newly added manifest file.

For Aligner, select from the following options:

BWA—Used for most input data.

TruSeq Amplicon Aligner—Used for TruSeq Amplicon data.

For more information on aligner options, see Alignment on page 1.

For Variant Caller, select from the following options:

Germline—Used for most other nontumor sample types.

Somatic—Used for tumor samples.

If you selected the Somatic Variant Caller, enter a value 0.05–30 to define the Somatic Variant Frequency Threshold (Percentage).

The default value is 5. The LowVariantFreq filter is applied to variants with frequencies below the specified threshold. Lower threshold values can result in false positive variants.

Enter a value 10–10,000 to define the Variant Caller Depth Filter level.

The default value is 10. Variants with a caller depth below the specified value are marked as filtered. Lower filter values can result in more false positive variants passing filter.

[Optional] In the Genotypes of Interest VCF field, select Select Dataset File(s).

Select at least one VCF file containing variants of interest.

The input VCF file must include the eight mandatory VCF columns and the FORMAT and SAMPLE columns. Only the CHROM, POS, REF, and ALT columns must contain data. Columns without a value must contain a period (.). The app ignores these columns.

The following table provides an example of a genotypes of interest file (*.vcf or *.vcf.gz) with the minimum required information.

##fileformat=VCFv4.1
#CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	SAMPLE
chr2	177016728	.	T	C	.	.	.	.	.
chr15	38545390	.	A	C	.	.	.	.	.
chr15	38545390	.	A	G	.	.	.	.	.
chr22	30090721	.	T	.	.	.	.	.	.

Set the Sample Identification Analysis option.

Generate sample fingerprints based on SNPs in the Sample ID panel spike-in to detect sample swaps. Sample identification is automatically disabled for nonhuman genomes.

For Indel Realignment, select from the following options:

On—Gemini performs indel realignment, which might improve medium-sized indel detection. However, overall accuracy can vary in different panels and total analysis time can increase. This option is the default setting.

Off—Indel realignment is not performed.

For Annotation, select from the following options.

RefSeq—Variants are annotated using RefSeq transcripts.

Ensembl—Variants are annotated using Ensembl transcripts.

None—Variants are not annotated.

Variant annotation is supported for human genomes only, and not supported for custom genomes.

Select Launch Application to start the analysis.

When the analysis is complete, the status of the app session is automatically updated and you receive a confirmation email.

For Research Use Only. Not for use in diagnostic procedures.