Detecting structural variations in NextGENe
If you select Detect structural variations, then NextGENe does the following:
1. Detects pseudo pair settings: NextGENe uses the settings that are specified in the Detect Pseudo pair pane of the Structural Variation Settings dialog box to determine the reads that are to be split as pseudo pairs by using the 3’ read “as-is” and reversing the 5’ end of the read.
Option | Description |
|---|---|
Detect Pseudo pair - NextGENe uses the settings that are specified in this pane to determine which reads are to be split in to pseudo pairs. | |
Mismatch ratio | Selected by default. The ratio of the number of mismatched bases for a read to the length of the read must be greater than the specified value. The default value is 0.3. |
Mismatch bases | Selected by default. The number of base pairs (length) of the mismatched bases for the read must be greater than the specified value. The default value is 50. |
Breakpoint filters - NextGENe uses the settings that are specified in this pane to determine which consensus sequences are to have their Linked Reads initially included in the Structural Variation report. All the specified criteria must be met for inclusion. See Structural Variation Report. | |
Coverage | At the suspected break points, the sum of the Linked Reads count + the Normal read count must be greater than or equal to the specified value. The default value is 5. |
Linked Reads ratio | The ratio of the Linked Reads count to the Total Coverage must be greater than or equal to the specified value. The default value is 0.001. |
Linked Reads | The Linked Reads count must be greater than or equal to the specified value. The default value is 3. |
Prioritize alignments to gene | Selected by default. An alignment score that is used in the underlying algorithm is increased if the alignment is to a region with gene annotation. Additional weight is given to the score if the alignment is to a region with gene annotation that is specified in a text file. To recognize this gene text file, click Specify gene list, and then click Load to browse to and select the text file. Each gene must be listed on a separate line in the text file. Example Gene list  |
2. Realign pseudo reads: NextGENe carries out an ordered series of steps to realign the pseudo reads.
a. NextGENe groups the overlapping reads that span a possible break point into a pair of consensus sequences. NextGENe then aligns the consensus sequences as well as the pseudo reads from which they were made to the reference. If any reads map to the RNA portion of the reference, then NextGENe remaps these reads to the DNA portion of the reference.
b. NextGENe classifies pseudo paired reads of consensus sequences where both ends map to the reference as Linked Reads and discards pseudo paired reads of consensus sequences where only one end maps to the reference.
c. NextGENe checks the matching percentage, coverage, Linked Reads count, and the Linked Reads ratio at the possible breakpoints, and then saves the Linked Reads as a Structural Variation event if all the criteria that is specified for the Breakpoint filters are met.