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Set Analysis Parameters

In BaseSpace, select the Apps tab.

Select TruSeq Amplicon.

From the drop-down list, select version 3.0, and then select Launch to open the app.

In the Analysis Name field on the app input form, enter the analysis name.

By default, the analysis name includes the app name, followed by the date and time that the analysis session starts.

From the Save Results To field, select the project that stores the app results.

From the Sample(s) field, browse to the sample you want to analyze and select the checkbox. You can select multiple samples.

[Optional] From the Custom Genome (Optional) field, browse to the FASTA file you want to use as the custom genome. You can select only one file.

From the Targeted Amplicons field, select a panel of targeted amplicons representative of the selected samples.

If you selected Custom Panel in the Targeted Amplicons drop-down list, upload a custom manifest and select the manifest file from the Custom Manifest File field. Upload a custom manifest as follows.

Navigate to your project in BaseSpace.

Select Import.

Follow the instructions to add the Custom Amplicon manifest file (*.txt) to the project.

Note

Specify the reference genome in the header of the manifest file. See Reference Genomes.

From the Variant Caller field, select a variant caller.

For tumor samples, use the Somatic Variant Caller.

If using the somatic variant caller, specify the Somatic Variant Caller Threshold (percentage), which is an integer (1–30). Set to 5 by default. Variants with a low frequency below the specified threshold get a LowVariantFreq filter. Lower threshold values can result in false positive variants.

Specify the Variant Caller Depth Filter level, below which variants are marked as filtered.

This is an integer (10–10000) that is set to 20 by default. Low filter values may result in more false positive variants passing filter.

[Optional] From the Genotypes of Interest VCF (Optional) field, select one or more VCF files containing variants of interest.

The input VCF must include the 8 mandatory VCF columns as well as the FORMAT and SAMPLE columns. The only columns that must be populated with data are CHROM, POS, REF, and ALT. Columns that do not have a value should be set to “.”; these columns are ignored.

The following table shows an example of a Genotypes of Interest VCF input file that includes 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 Read Stitching option.

When enabled, reads that overlap > 10 bases between Read 1 and Read 2 are combined to create a single (longer) read for alignment.

From the Annotation field, select a preferred gene and transcript annotation reference database.

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

Select Continue.

The TruSeq Amplicon App begins analysis of the samples.

When analysis is complete, the status of the app session is updated automatically and an email is sent to notify you.

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