This is an example analysis of pairwise alignments using PanGEA. The ESTs used in this example are from the publication of Torres et al. (2008) and have been mapped to the genes of D. melanogaster. The analyis screen consists of three main parts: the text box containing 'General information', the text box containing the analysis 'Results' and the area on the right-hand side containing the options and parameters for the users.
| ambiguous | use ambiguously mapped ESTs, unambiguously mapped ESTs or both. Whether an EST is considered ambiguous does not depend on the input-file, it depends on how often a query-sequence ID (EST-ID) is in summary found in the input files. An unambigously mapped EST should of course occure only once. |
| min. alignment | minimum length of the alignment, not considering introns |
| min. EST aligned | how much of an EST has at least to be aligned. Value is specified in percent of the EST length. |
| min. similarity | minimum similarity of the query sequence with the database sequence in percent. Indels are not considered. |
| min. tags per gene | minimum number of ESTs mapping to a gene |
| sense-antisense | display sense-transcripts, antisense-transcripts or both. If the EST could be directly mapped, it accounts as a sense-EST, if the reverse complement of the EST could be mapped it accounts as antisense-EST |
| min. gap length | minimum length for a gap as to be considered a long gap. |
| consider gaps | display only alignments having long gaps, or alignments having no long gaps or both. |
| gap position | display only alignments having long gaps in the database sequence, or in the query sequence or in both. In case the alignment contains a long gap may be an intron. |
| update analysis | update the analysis using the parameters specified by the user |
| export subset | export the content of the active subset, i.e the user-specified subset |
| clipboard | Copy the content of the 'Results' text box into the clipboard |
This text box contains some general information, such as the total number of ESTs loaded, the number of subset ESTs, i.e ESTs meeting the user-specified subset requirements. It also displays how many genes are covered by all ESTs and the subset-ESTs. With the settings applied in the example shown below, only 229 genes are covered with the subset-ESTs from an initial of 3739.
Example:
PanGEA Pairwise Alignment and Transcriptome Analysis- 4/27/2008 4:41:14 PM
Total tags loaded 22713
Total genes loaded 3739
Subset tags selected 12459 (automatically selected for export)
Subset genes selected 229
Active settings
Show unambiguous tags True; show ambiguous tags False
Minimum length of alignment 60; minimum similarity of alignment 98.00
Minimum coverage of tags (percent) 90.00; minimum frequency of tags 10
Display sense tags yes; display antisense tags yes
Pairwise nucleotide sequence files loaded
Pub_Torres_2008.aln
In the results, summary statistics are shown for each gene-ID. For example:
| FBgn0013686 | 5572 | 5287 | 80.20 | 424.35 | 0 | 0 | 0 |
| FBgn0013674 | 358 | 323 | 98.45 | 25.93 | 0 | 0 | 0 |
| FBgn0040007 | 55 | 44 | 100.00 | 3.53 | 32 | 29 | 3 |
| a | b | c | d | e | f | g | h |
| a | The ID of the database sequence, gene or chromosome |
| b | The total number of ESTs mapping to this database sequence |
| c | The number of subset-ESTs mapping to this database sequence |
| d | The number of sense-ESTs mapping to this database sequence in percent. This value is calculated for the active subset. |
| e | relative expression level in promille. The number of subset-ESTs mapping to this gene divided by all subset-ESTs (sum for each gene) multiplied with 1000. |
| f | the number of long gaps found in the subset-ESTs mapping to this gene. This is the sum of 'g' and 'h'. The threshold for gaps to be considered long gaps has to be specified by the user. |
| g | the number of long gaps in the database sequence (putative introns) |
| h | the number of long gaps in the query sequence (structural variations) |
Torres TT, Metta M, Ottenwälder B, Schlötterer C.
Gene expression profiling by massively parallel sequencing.
Genome Res. 2008 Jan;18(1):172-7. Epub 2007 Nov 21.