Genomic Project Input Sub-Tab

2.7.1

The Input sub-tab for genomic projects is shown on Figure 56 (for cDNA projects, see Section 2.7.4). It allows you to adjust the following settings:

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When the Nimblegen sequence capture checkbox is checked, the read data will automatically be primer-trimmed assuming NimbleGen Sequence Capture adapters are present at the beginning of the reads (this option defaults to unchecked). If the NimbleGen gSel protocol has been used, this option must be checked. There is no need to use this option with NimbleGen’s optimized protocol for the GS FLX Titanium chemistry.

NOTE on trimming in general: new reads added to a project are trimmed when the mapping computation is performed. The trimming is performed using the parameter settings on the Project Input sub-tab at the time the computation is run. Reads for which the mapping computation has already been run are not re-trimmed.

•	If the Automatic trimming checkbox is checked, the ends of new read files added to the project will be trimmed the next time a mapping computation is performed.

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Expected depth allows you to specify the number of reads expected to uniquely map to a single position of the reference genome. For high-depth mapping (where the expected coverage of a position in the genome could reach hundreds or thousands of reads), this option can be used to prevent low-frequency variations from being reported. If this option isn’t used, many variations that are actually due to sequencing errors may be reported. The default value for this option is 0. A value of 0 or greater is allowed. If this option is set to a very low value (below 25), any variations that appear in only two reads may be output to the 454AllDiffs.txt file.

•	The Minimum Read Length option can be used to change the minimum accepted read length to be used in the mapping (default is 20 bp, allowed value range is 15-45). This option is only applied if Paired End data is used.

The input files section of the tab allows you to specify the locations of several files that may be used in the mapping.

•	An optional Trimming database is used to trim the ends of input reads (for cloning vectors, primers, adapters or other end sequences). Specify the path to a FASTA file of sequences to be used for this trimming (see Section 4.10).

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To use the Screening database option, set the path to a FASTA file of sequences to be used to screen the input reads for contaminants. A read that almost completely aligns against a sequence in the screening database is removed so that it is not used in the computation; if at least 15 bases of a read do not align to the screening sequence, no action is taken (see Section 4.10).

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The Targeted regions option can be used to direct the GS Reference Mapper to only report output for regions of the reference you specify and the reads / contigs that overlap those regions. The reads are mapped against the complete reference, but the output files will only report on the specified regions. See Section 4.17 for more details on target regions and extended target regions. The string value can be

◦	a “accno:#-#” string, e.g. “chr9:10549-30594” specifies bases 10549 to 30594 of the chr9 reference sequence

◦	the path to a file containing lines of “accno:#-#” strings (one per line)

◦	the path to a GFF file created during a NimbleGen Sequence Capture experiment, describing the “primary_target_region” regions of the experiment. See appendix 4.10.1 for details of the GFF file format.

This option may is useful when performing SeqCap or CHiPSeq projects.

•	If you are performing a mapping against an annotated reference, you may have to specify the locations of the annotation files (unless a GoldenPath reference is being used)

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To use the Genome annotation option, enter the path to an annotation file describing the gene/coding-region annotations for the reference sequences, so that the variation detection (AllDiffs, HCDiffs, and Structural Variations) can report gene names and protein translations of any identified variations in gene regions. The format of this file must match that of the GoldenPath “refGene.txt” file. See appendix 4.10.2 for details of the refGene.txt file format.

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The Known SNP option is used to specify the path to an annotation file describing the known SNP information for the reference sequences, so that the variation detection (HCDiffs tab) can link identified variations to the known SNP information. The format of this file must match that of the GoldenPath snp130.txt file. See appendix 4.10.3 for details of the snp130.txt file format.

•	The Accno Renaming File option can be used to specify a renaming file to incorporate gene and transcript descriptions into the output files and/or to provide more meaningful names for genes and transcripts. For a description of the renaming file see Section 4.9

The software does three things if no Genome annotations or known SNP paths are specified:

•	If there is a single reference file, the software looks for a file with the same name as the reference, but with a .refGene/.refLink/.snp* suffix as the annotations.

•	If the reference file(s) is (are all) in the same directory, the software looks in that directory for a “refGene.txt”, “refLink.txt” or “snp*.txt” file for the annotation files.

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If the GOLDENPATH environment variable is set for a genomic mapping project, and the reference files are in a “chromosomes” directory inside the GOLDENPATH tree, the software looks for a “database” directory at the same level as the “chromosomes” directory, and then looks for the annotation files inside the database directory. As a consequence the situation may arise whereby a project gets annotated even if no annotation file has been specified in the parameters tab.

•	The Include and exclude filter file options can be used to specify a file of sequences to specifically include or exclude in the computation. The file format and functionality are the same as the –e and –i options of sfffile (see Section 3.1).

Long file paths will be displayed in full when you hover the mouse cursor over the text area.