Deep Sequencing Data Analysis

Example use: Basic analysis of deep sequencing data

Offline HCV-GLUE can be used for minority variant analysis of deep sequencing data in the form of SAM/BAM files We presume here that a suitable sequence assembly method has been used to generate the SAM/BAM file from the raw sequencing data.

We will use the phdrSamReporter module within HCV-GLUE to translate those reads within the file which map to the NS5A gene. The function will report the balance of different amino acids residues at different locations within this gene.

Enter the GLUE command below and check the output. Use 'Q' to exit the interactive table.

GLUE> module phdrSamReporter amino-acid -i ngsData.bam -r REF_MASTER_NC_004102 -f NS5A -p -a AL_UNCONSTRAINED
+============+==========+==========+===========+=============+============+
| codonLabel | samRefNt | relRefNt | aminoAcid | readsWithAA | pctAaReads |
+============+==========+==========+===========+=============+============+
| 1          | 6147     | 6258     | Y         | 1           | 0.32       |
| 1          | 6147     | 6258     | S         | 314         | 99.37      |
| 1          | 6147     | 6258     | P         | 1           | 0.32       |
| 2          | 6150     | 6261     | S         | 1           | 0.32       |
| 2          | 6150     | 6261     | R         | 1           | 0.32       |
| 2          | 6150     | 6261     | G         | 313         | 99.37      |
| 3          | 6153     | 6264     | S         | 315         | 100.00     |
| 4          | 6156     | 6267     | W         | 315         | 100.00     |
| 5          | 6159     | 6270     | L         | 314         | 100.00     |
| 6          | 6162     | 6273     | R         | 312         | 99.36      |
| 6          | 6162     | 6273     | K         | 1           | 0.32       |
| 6          | 6162     | 6273     | G         | 1           | 0.32       |
| 7          | 6165     | 6276     | D         | 314         | 100.00     |
| 8          | 6168     | 6279     | I         | 274         | 100.00     |
| 9          | 6171     | 6282     | W         | 274         | 100.00     |
| 10         | 6174     | 6285     | D         | 253         | 100.00     |
| 11         | 6177     | 6288     | *         | 1           | 0.40       |
| 11         | 6177     | 6288     | W         | 252         | 99.60      |
| 12         | 6180     | 6291     | I         | 197         | 100.00     |
+============+==========+==========+===========+=============+============+
Rows 1 to 19 of 753 [F:first, L:last, P:prev, N:next, Q:quit]

Example use: Combined genotyping and drug resistance analysis reports

Offline HCV-GLUE contains modules which run combined genotyping and drug resistance analysis procedures, and generate detailed reports.

This first example will read in the FASTA file, resistanceGeno1.fasta and produce an HTML report file resistanceGeno1.html. Click here for a preview of the report.

Mode path: /project/hcv
GLUE> module phdrReportingController invoke-function reportFastaAsHtml resistanceGeno1.fasta resistanceGeno1.html

The second example will read in the BAM file, ngsData.bam and produce a new HTML report ngsData.html, using a 15% reads percentage minimum threshold for reporting polymorphisms. Click here for a preview of the report.

Mode path: /project/hcv
GLUE> module phdrReportingController invoke-function reportBamAsHtml ngsData.bam 15.0 ngsData.html

HCV-GLUE can also generate files containing genotyping and drug resistance reporting data in a machine-readable format (XML or JSON). These could be used e.g. for integration into a bioinformatics pipeline. Contact the team if this is of interest.

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