Folder/filename customization is REQUIRED:

$MyDisk and $Folder define the location (folder or subdirectory) of the input and output files and must be customized by replacing the placeholder names (e.g. DISKNAME) with the names on your computer system. Look for this banner on the first page of BlastReport to find the variables that must be modified:

Note: Various operating systems use different characters between folder names or between a directory and its subdirectories in a full file name. Make sure to include this character in the $Folder literal (characters between the quotes,"). The $Folder and $MyDisk literals must end with this character.

For Unix, directories are connected by a forwardslash '/' /HomeDirectory/SubDirectory/MyFile

Perl uses a backslash '\' as an escape character for special uses such as \n for new line. Use \\ instead of a single backslash in Windows file names. e.g. $MyDisk = "c:\\"; will work correctly

----------------------------------------------------------------------------

The remaining file and folder name variables can be used to change the names of the files that are read and written by BlastReport. For input, BlastReport reads the directory as defined above and looks for files that start with the literal defined by $BlastPre. Then select the dataset that you wish to analyze. This requires the files written by blastcl3 to start with this prefix ($BlastPre value).

Optional parameters to change report/file names:

These variables control the amount of data that is printed. Look for the following text to find these variables in BlastReport. These variables follow the ones for file and folder names.

# ---------------------------------------------------------------------------

# C H A N G E P R I N T C O N T R O L P A R A M E T E R S H E R E:

# ---------------------------------------------------------------------------

The print control variables are limits on the amount of information to be printed in the reports generated by BlastReport. The default values set here are limits that we have found useful for our work. You may want to change these to optimize the reports for your use. These merely put limits on the amount of data to be printed in the reports and are not 'intelligent' settings. Increasing some of the settings to very high values may result in a an 'out of memory' error, especially if you are using a large number of query sequences.

There are 4 groups of print variables and 1 single control variable. For the groups, there is a 'master' variable followed by a recommended default value for markers and cDNAs. The first or 'master' variable in each group should not be modified. The master variables are set at execution time as indicated by your response to:

Select type of Input:

1 Marker

2 cDNA

>Enter number for type of file or Q to quit:

Running Perl differs by operating system. Unix and Windows both run from the command line, Perl for Windows runs in a DOS window. On MacOS, there is a GUI interface.

Unix and Windows(in a DOS window) on the command line type: perl BlastReport2

MacOS: Start perl by double clicking on the macperl icon. Click Script on the menu bar and select Run Script. Select BlastReport2 from the folder where it was saved. (Double click or Open).

Once started, BlastReport2 runs similarly on all operating systems.

The execution of BlastReport requires 3 inputs:

1. The chromosome number or whatever number that you have chosen. If you enter 4, it will prefix with a zero to use '04'.
2. The number of the file to be analyzed. A list of choices will be presented.
3. The type of input: marker or cDNA. See discussion below under print customization for an explanation of how 'marker' and 'cDNA' responses modify the reports written by BlastReport.

Sample execution of BlastReport2:

==== Begin BlastReport Parameter Selection ====

>Enter chromosome number of blast report to use or Q to quit.

5

blast 05 files:

1 blast 05 09-05 cDNA

2 blast 05 AC021079 04-19

3 blast 05 gabra1 04-19

4 blast 05 gene nr 04-18

5 blast 05 genes 04-18

6 blast 05 ht 03-29

>Enter number of file to use or Q to quit: 1

Using file: blast 05 09-05 cDNA

Select type of Input:

1 Marker

2 cDNA

>Enter number for type of file or Q to quit: 2

Type of file selected: cDNA

==== Begin BlastReport Execution ====

INFO: BLASTIN opened successfully:

IMac03:Folder:blasts:blast 05 09-05 cDNA

INFO: Number of Query Sequences in the run: 18

INFO: Number of Query Sequences with alignments: 16

INFO: Sequences that may have significant alignments: 16

INFO: Full Report: IMac03:Folder:blasts:BRept_05_09-05_cDNA_Full

INFO: Summary: IMac03:Folder:blasts:BRept_05_09-05_cDNA_Sum

INFO: Excel Table: IMac03:Folder:blasts:BRept_05_09-05_cDNA_Tab

==== BlastReport Execution Completed ====

BlastReport reads the output of a blastcl3 query, which contains the search results of one or more query sequences. BlastReport searches the blastcl3 output for alignments and generates 2 reports, Full and Sum, and a tab-delimited file (Tab).

To describe the features of BlastReport output, sample output is available on this website. These reports were generated on an iMac, but results on Unix or Windows would be very similar if not identical. Three microsatellite markers were taken at random for human chromosome 17, D17S1868, D17S1298 and D17S1308. A text file containing these 3 sequences in FastA format were used as input to blastcl3, using the following parameters: program (blastn), database(htgs), expectation(1e-30), filtering was turned off (F), descriptions(10), alignments(10), output (blast 17 09-15 mrker). These marker were chosen because they are good examples of what may be seen.

The reports produced by this execution of BlastReport1.0 are:

BRept_17_09-15_mrker_Full

BRept_17_09-15_mrker_Sum

BRept_17_09-15_mrker_Tab

--- D17S1308 alignment analysis ---

Number of alignments: 144

Region(s) of alignment for Query: D17S1308 (first 12 only)

From: 1 To: 636 Expect: 0.0 Match: 602/636 AC008087.3

From: 148 To: 318 Expect: 4e-38 Match: 148/171 AC008087.3

From: 185 To: 316 Expect: 1e-34 Match: 117/132 AC008087.3

From: 180 To: 288 Expect: 1e-34 Match: 100/109 AC008087.3

From: 154 To: 315 Expect: 9e-33 Match: 139/162 AC008087.3

From: 1 To: 636 Expect: 0.0 Match: 602/636 AC025743.2

From: 148 To: 300 Expect: 2e-46 Match: 138/153 AC025743.2

From: 158 To: 288 Expect: 4e-38 Match: 118/131 AC025743.2

From: 148 To: 288 Expect: 6e-37 Match: 125/141 AC025743.2

From: 148 To: 316 Expect: 6e-37 Match: 146/169 AC025743.2

From: 180 To: 301 Expect: 4e-35 Match: 110/122 AC025743.2

From: 180 To: 288 Expect: 1e-34 Match: 100/109 AC025743.2

The first line, (A) is an 'eye catcher' that makes it easy to scan for the beginning of the information for a query. Or you may use a word processor to find '---===' which is unique to this line. Next is the query description line as listed by BLAST, (B). Immediately following that are the descriptions of database sequences that align to this query, (C). The number of these descriptions is limited by the -v parameter in the blastcl3 execution. The next section (D), is information extracted from all of the alignments for this sequence. It gives the total number of alignments found for this query in this case 144. Regions of alignment for your query sequence are given next, $MaxAlign displayed is limited by the $MaxAlign value, in this case the value is 12. (See Print Customization above.) The first alignments regions are given. For each the region of your query in the local alignment, From: /To:, the expectation for this alignment, the quality of the match is given by a ration of identical nucleotides to total number of nucleotides in the local alignment. The first local alignment spans the entire length of the query, 1-636, has an expectation of 0.0 and 602 out of 636 nucleotides match. The database sequence that gives this alignment is AC008087.3. The next 4 local alignments only span part of D17S1308, in the area of 148 to 320, this is an indication that this portion of D17S1308 is a highly repeated sequence, such as an ALU or Line sequence. The fact that there were 144 total local alignments for these 10 database sequences reinforces this interpretation. In fact if no limits are put on the number of descriptions returned, D17S1308 has thousands of alignments with and expectation better than 1e-30. This is one reason to limit the number of descriptions and alignments when running blastcl3, -v and -b respectively. There is one more significant alignment in this group for AC025743.2. Notice the expectation for given in the description line for AL121672.10, this may also be a significant alignment, but no more information is given for it to conserve space. This is an example of when you may want to increase the value for $MaxAlign, specifically $MarkerAlign, and rerun the BlastReport. Alternately, you may view the original blastcl3 output to see to alignment. Checking the information in the next section can also help: (some modification to fit page).

>>>>>>> ---------------------------------------- <<<<<<<<

D17S1308 may have a significant alignment.

D17S1308 1 - 636 148 - 318 185 - 316 180 - 288

AC008087.3 35170 - 35784 4707 - 4877 89055 - 88924 48450 - 48558

D17S1308 1 - 636 148 - 300 158 - 288 148 - 288

AC025743.2 58951 - 59566 42245 - 42397 72684 - 72814 34561 - 34701

D17S1308 1 - 361 347 - 636 158 - 291 148 - 318

AL121672.10 141032 - 141388 141362 - 141645 17300 - 17433 118768 - 118938

D17S1308 148 - 317 148 - 316 180 - 318 186 - 316

AC016560.7 151389 - 151220 33912 - 34080 23265 - 23402 85425 - 85295

This section starts with another eye-catcher. For the first 4 database sequences that have alignments, the first 4 regions of alignment are given, see limits $DBseqPrint and $RegionPrint above. For database sequence AL121672.10, the first 2 regions of alignment span the length of the query sequence. This is a case where the length of tandem repeat segments for the marker sequence and the genomic sequence vary significantly. So 2 local alignments are seen instead of 1. You can see this by examining the nucleotide numbers for the database sequence, AL121672.10 and noticing that the 2 local alignments overlap. Viewing the full alignments in the original blastcl3 will confirm this. (See query D17S1298 for another example of this. In this case the alignment is available to view in BlastReport1.0.) For Database sequence, AC016560, the only alignments are in the region of the repetitive segment. D17S1868 shows the usefulness of the print limits. The original blastcl3 report is 58 pages long, most of it (53 pages) are alignments to this repeated region. In some cases, the expectation limit will eliminate these repetitive alignments from the blastcl3 report. But these alignments are good enough and long enough to be included. The conservative print limits eliminated a useful alignment in the first section, but the regions of alignment in the 2^nd section lists the alignment so that you can determine if you need to examine this further. Note that the description for AL121672.10 states that it is a chromosome 22 sequence. We have found several sequences in htgs that have been mislabeled. Don't discard a sequence based on the description. Use the sequence to search htgs and see if it aligns with other sequences labeled chr 17 or chr 22.

The last section of the report for a query contains the first four full alignments for this query sequence. (See $MaxFull above for this limit.)

The tab file contains the query sequence, the database sequence that it aligns to, the name of the clone if given, the length of the database sequence, the expectation of the best alignment, and last the alignment regions for the best alignment. Lalign1 and Lalign2, give the begin and end sequence numbers for the query sequence (or locus). Salign1 and Salign2 give the begin and end numbers for the alignment for the database sequence.