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User guide


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

ACGTRead depth
Minor allele percentageComments
000440%No call, since read depth below 5
000550%Homozygous T
1004520%Heterozygous A/T, as minor allele is 20% of major+minor allele read depth
1103425%No call, as read depth of major plus a single minor allele is below 5
14171136%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)
01009010010%Heterozygous A/T, since minor allele is 10% of major+minor allele read depth
0100911019.9%Homozygous T, since minor allele is <10% of major+minor allele read depth
0100899910.1%Heterozygous A/T, since minor allele is >10% of major+minor allele read depth
01049010010%Heterozygous A/T, since minor allele is 10% of major+minor read depth

Table 1

Creating an annotation file

AgileAnnotator Screenshot 1

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 Create button in the Create annotated feature file panel (Figure 1). This causes the Annotation file creation window to be displayed (Figure 2).

AgileAnnotator Screenshot 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 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 Chromosomes 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 press the CCDS 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. These files can be downloaded from the NCBI CCDS web page or FTP site.

Finally, press the Create 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

AgileAnnotator Screenshot 3

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 Type of quality score option, and and to adjust the variant calling cut-off parameters (quality and read depth), all accessible under the Options menu (Figure 3).

AgileAnnotator Screenshot 4

Figure 4: Entering data

Once the variant calling options have been set, load the genome annotation file by pressing the Select 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 Limit by 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.

AgileAnnotator Screenshot 5

Figure 5: File formats used to limit the regions screened for sequence variants

AgileAnnotator Screenshot 6

Figure 6: The title bar of AgileAnnotator shows the status of the analysis

Next, choose an ORDERED SAM file using the Select 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 Screen 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 AgileVariantViewer can be used to view the variant data.

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