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Set Analysis Parameters

Set Analysis Parameters

1. Open the RNA-Seq Alignment app from BaseSpace Sequence Hub as follows.
  1. Select the Apps tab, and then select RNA-Seq Alignment.
  2. From the Version drop-down list, select 2.0.0.
  3. Select Launch Application.
2. 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..

3. From the Save Results To field, select Select Project.
4. In the Select Project dialog box, select where to store app results, and then select Select.
5. From the Biosample(s) field, select the biosample you want to analyze as follows.
  1. Select Select Biosample(s).
  2. Select the checkbox of each biosample you want to analyze, and then select Select.
  3. Select the Strandedness for each sample as follows.
    • First Strand–If the samples were prepared with a TruSeq Stranded Total RNA Sample Prep Kit, TruSeq Stranded mRNA Sample Prep Kit, or another RNA library prep kit that produces reads in the orientation of the first-strand cDNA.
    • Second Strand–If the samples were prepared with a ScriptSeq v2 RNA-Seq Library Prep Kit or another kit producing reads in the opposite direction of the first-strand cDNA.
    • No–If the samples were prepared with an unstranded library prep kit.

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.

6. From the Reference Genome drop-down list, select the reference genome to be used for alignment.

The default is Homo sapiens (PAR-masked)/hg19 (RefSeq).

7. [Optional] If you selected Custom from Reference Genome, select Select Datasets, and then select a custom genome.

Only custom genomes created by the RNA Custom Genome Builder app are supported.

8. If this sample was generated using a targeted enrichment panel, select the panel from the Panel drop-down list. Otherwise, select None.
9. [Optional] Select the QC Mode checkbox to analyze only the subset of a read pairs for each sample. If selected, enter the number of read pairs to be analyzed from each sample.
10. [Optional] Select the Call Fusions checkbox to enable fusion calling.

By default, the checkbox is selected if you selected a panel that supports fusion calling. Paired-end reads are required.

11. If the sample includes ERCC RNA spike-in controls, select the appropriate mix from the the ERCC Spike-In Controls drop-down list. Otherwise, select None.
12. [Optional] To force genotype a set of candidate variants, select Select Dataset File(s) and select up to five VCF files 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.

NOTE

The variant of interest VCF file must be a valid variant-only VCF (not genome VCF) in which all records are left-normalized. See Strelka.

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
.
.
.
.
.
.
13. [Optional] Select the Set Advanced Options checkbox to enable the advanced options and then specify the values for the appropriate options. See Advanced Options.
14. Select Launch Application.

The RNA-Seq Alignment App begins analysis.

When analysis is complete, the app updates the status of the session and sends you an email notification.