User guide
Introduction
AgileAnnotator identifies sequences that have been aligned to the protein-coding sequences
or to regions within 50 bp of a splice junction. Each read is then screened for sequence mismatches and insertions or deletions
highlighted in its corresponding “CIGAR” data field in the SAM file. A position is called only if at least 5 reads
for the two most common alleles are mapped to the position. A position is called as heterozygous if 10% or more of the reads
identify the minor allele. The variant-calling algorithm is designed to be promiscuous, since the final cut-off parameters are
set interactively later, using AgileVariantViewer.
Table 1 illustrates the variant-calling process:
File formats
A description of the file formats for the variant and read depth files created by AgileAnnotator can be found here.
Variant calling examples
| A | C | G | T | Read depth (major+minor) | Minor allele percentage | Comments |
|---|---|---|---|---|---|---|
| 0 | 0 | 0 | 4 | 4 | 0% | No call, since read depth below 5 |
| 0 | 0 | 0 | 5 | 5 | 0% | Homozygous T |
| 1 | 0 | 0 | 4 | 5 | 20% | Heterozygous A/T, as minor allele is 20% of major+minor allele read depth |
| 1 | 1 | 0 | 3 | 4 | 25% | No call, as read depth of major plus a single minor allele is below 5 |
| 1 | 4 | 1 | 7 | 11 | 36% | No call, as minor allele read depth is not more than twice the combined read depth of the two remaining (presumably erroneous) bases (A+G) |
| 0 | 10 | 0 | 90 | 100 | 10% | Heterozygous A/T, since minor allele is 10% of major+minor allele read depth |
| 0 | 10 | 0 | 91 | 101 | 9.9% | Homozygous T, since minor allele is <10% of major+minor allele read depth |
| 0 | 10 | 0 | 89 | 99 | 10.1% | Heterozygous A/T, since minor allele is >10% of major+minor allele read depth |
| 0 | 10 | 4 | 90 | 100 | 10% | Heterozygous A/T, since minor allele is 10% of major+minor read depth |
Table 1
Creating an annotation file
Figure 1: AgileVariantViewer user interface
AgileAnnotator is designed to identify sequence variants that can be suspected to disrupt a protein’ function. It therefore only considers sequence variants that are within or close to protein-coding sequence.
To achieve this, AgileAnnotator makes use of an annotation file that contains the locations of protein-coding exons and their genomic sequences. This file can be created either by AgileAnnotator or by AgileGenotyper. To create an annotation file, first press the button in the Create annotated feature file panel (Figure 1). This causes the Annotation file creation window to be displayed (Figure 2).
Figure 2: The annotation creation window
Note: The genomic sequences in the annotation files are derived from the current genomic sequence reference files and MUST match the version used when aligning the sequence reads to the genome and they MUST also be the same build version used by the creators of the CCDS or the RefSeq data files, used to identify the positions of coding sequences. If different reference sequences are used the analysis will fail!
The location of the uncompressed FASTA-format genomic reference files is selected using the button under Chromosome sequence files. These reference sequence files must follow the specific naming convention where each file name starts with "chr" followed by either the chromosome number or "X" or "Y", and has the .fa file extension. Permitted names include chr1.fa, chr5.fa for an autosome, while the X and Y may be named chrX.fa or chr23.fa and chrY.fa or chr24.fa, respectively.
Next select the source of the gene annotation file (CCDS or RefSeq) and then press the button in the CCDS data files panel and select the file containing the positions of the coding sequences as described by the Consensus CDS (CCDS) project or RefSeq datasets. The CCDS file can be downloaded from the NCBI CCDS web page or FTP site. While the RefSeq data file can be obtained from the UCSC Genome Browser's Tables page as described here.
Finally, press the button under Create annotation file and enter a name for the genomic annotation file. Since AgileAnnotator has to read all of the sequences in the genomic reference files and then write a large amount of data, the creation of the annotation file may take several minutes.
Screening a SAM file for sequence variants
Figure 3: Adjusting the variant calling parameters
Before a SAM file is screened for sequence variants, it is necessary to select Solexa- or Sanger-type quality scores, using the option, and and to adjust the variant calling cut-off parameters (quality and read depth), all accessible under the menu (Figure 3).
Figure 4: Entering data
Once the variant calling options have been set, load the genome annotation file by pressing the button in the Genome annotation file panel (Figure 4). Since this file is large, it may take a while to load. It is VERY important that the annotation file was created using the same genomic reference sequence files used to align the sequence data in the SAM file.
It is possible to limit the exported sequence variants to those originating from a specified list of genes, genomic regions or chromosomes. To do this, select the appropriate option in the Limit analysis panel and then press the button to choose a plain text file containing the data. Figure 5 shows the format of these files, with X and Y indicating the sex chromosomes. Gene names must match those in the CCDS file. To export variants from across the whole genome, create a chromosomal limits file containing the numbers 1 to 24.
Figure 5: File formats used to limit the regions screened for sequence variants
Figure 6: The title bar of AgileAnnotator shows the status of the analysis
Next, choose an ORDERED SAM file using the button in the Alignment file panel (Figure 4). The aligned sequence reads in this file must be ordered by chromosome and chromosomal position.
Finally, press the button in the Screen alignment data panel (Figure 6) and enter the name of the file to save the exported sequence variants to. AgileAnnotator will then export the sequence variants as it reads the SAM file, showing the progress of the analysis in the window title bar. AgileAnnotator will also generate a second file that contains read depth information for each of the exons that sequence reads were mapped to. The latter file is designed to be used by AgileVariantViewer. Since AgileAnnotator only stores the sequence reads for one gene at a time, it is does not require large amounts of memory, and the speed of the analysis is limited by the speed at which it can read the SAM file, which should therefore be on a local hard drive. A description of the variant and read depth file formats can be found here, while AgileVariantViewer and AgileFileViewer can be used to view the variant data.