Set Analysis Parameters
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1
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Open DNA Amplicon from BaseSpace™ Sequence Hub as follows. |
|
a
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Select the Apps tab, and then select DNA Amplicon. |
|
b
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From the Version drop-down list, select 2.0.0. |
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c
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Select Launch Application. |
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2
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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.
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3
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From the Save Results To field, select Select Project, and then select a project to store app results to. |
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4
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From the Biosample(s) field, select the biosample you want to analyze as follows. |
|
a
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Select Select Biosample(s). |
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b
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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.
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5
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[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. |
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6
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From the Targeted Amplicons drop-down list, select a panel of targeted amplicons representative of the selected samples. |
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7
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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. |
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b
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Select Projects from the My Data tab.
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|
c
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Select the project you want to upload the manifest file to. |
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d
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Select File | Upload | Files | Manifest. |
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e
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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. |
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f
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Return to the DNA Amplicon app and repeat step 7 to select the newly added manifest file. |
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8
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For Aligner, select from the following options: |
|
u
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BWA—Used for most input data. |
|
u
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TruSeq Amplicon Aligner—Used for TruSeq Amplicon data. |
For more information on aligner options, see Alignment on page 1.
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9
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For Variant Caller, select from the following options: |
|
u
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Germline—Used for most other nontumor sample types. |
|
u
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Somatic—Used for tumor samples. |
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10
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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.
|
11
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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.
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12
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[Optional] In the Genotypes of Interest VCF field, select Select Dataset File(s). |
|
13
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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
|
.
|
.
|
.
|
.
|
.
|
.
|
|
14
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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.
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15
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For Indel Realignment, select from the following options: |
|
u
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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. |
|
u
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Off—Indel realignment is not performed. |
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16
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For Annotation, select from the following options. |
|
u
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RefSeq—Variants are annotated using RefSeq transcripts. |
|
u
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Ensembl—Variants are annotated using Ensembl transcripts. |
|
u
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None—Variants are not annotated. |
Variant annotation is supported for human genomes only, and not supported for custom genomes.
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17
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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.
BaseSpace DNA Amplicon v2.0 App Online Help
For Research Use Only. Not for use in diagnostic procedures.