To parse barcoded sample files
1. On the NextGENe main menu, click Tools > Barcode Sorting.
The Barcode Sorting dialog box opens.
2. Specify the Barcode sorting for the samples files—Barcode in sequence, Barcode in read name, or Barcode in separate file.
The options on the Barcode Sorting dialog box are dynamically updated based on the Barcode sorting option that you select.
3. Click Add to browse to and select your sample files.
The sample files are listed by name in the Sample List pane. The name includes the full directory path to each sample file.
4. Select one of the following options:
Setting | Description |
|---|---|
Import index file | Select this option if you created a Barcode/Primer file with known barcode information. Click Import to browse to and select the Barcode/Primer file that you want to import, and then select one of the following: • Perfect Match—If you select this option, the tag for a read must be an identical match to the tag that is defined in the Barcode/Primer file, or the read is not allocated to the tag. • Loose Match—If you select this option, the tag for a read is divided into three equal segments—the first half, the second half, and the middle segment. Only one of these three segments must be an identical match to the tag in the Barcode/Primer file for the read to be allocated to the tag. Note: The Loose Match method is especially useful for longer tag sequences where the likelihood of sequencing errors within the tag region is greater. Note: If you are using Barcode Sorting in the NextGENe AutoRun tool, you must import an index file. |
Determine index automatically | Select this option if barcode information is not known and you want NextGENe to automatically the detect barcode information, and then do the following: • Indicate the barcode length. (Available only if you selected Barcode in Sequence.) • If you know the total number of true tags, select Total Number of Tags, and then enter the value. Note: When automatically detecting the number of true tags, the Barcode Sorting tool includes only the most frequently observed sequences to avoid parsing reads according tags that are the result of sequencing errors. Note: This option is not applicable for the NextGENe AutoRun tool. |
5. Depending on your Barcode sorting selection, do the following:
Barcode Sorting Selection | Options |
|---|---|
Barcode in sequence | 1. If the forward read and the reverse read each have their own barcodes, then select Dual index. 2. Continue to Step 6. |
Barcode in read name | N/A.Continue to Step 8. |
Barcode in separate file | 1. Select one or both of the following: • Paired Reads: If you are loading paired read data, then select Paired Reads. • Dual Index: If the forward read and the reverse read each have their own barcodes, then select Dual index. 2. Continue to Step 8. |
6. Optionally, click Advanced Settings to open the Advanced Settings dialog box and select the appropriate settings for your data; otherwise, go to Step 8.
Setting | Description |
|---|---|
Dual-Barcode | Automatically selected and unavailable only if Dual Index has been selected; otherwise, not selected and unavailable. |
Barcodes at 5’ End Only | Selected by default and available only if Dual Index is not selected. Checks for barcodes only at the 5’end of reads. |
Check Reverse Complements of Barcodes | Selected by default. This option allows for any of the following four tag combinations: • Forward + Forward • Reverse + Reverse • Forward + Reverse • Reverse + Forward Clear this option if do not want NextGENe to check for the reverse complements of barcodes. |
454 Sample orientation estimation - Estimate sample orientation before sorting | Applicable only for Roche/454 data and available for selection only if the following two conditions are met: • Barcode in sequence is selected. • Import index file is selected. After selecting this option, click Load to load a .gbk or .fasta reference file, or click Preloaded to select a preloaded reference. This results in the alignment of the reads being carried out against the reference before barcode sorting is carried out. |
7. Click OK.
The Advanced Settings dialog box closes and you return to the Barcode Sorting dialog box.
8. In the Output pane, do the following:
• If you selected Barcode in sequence and the reads in the output file are to include the barcode sequences, then select “Keep the barcode in the sequences.”
• Leave the default value for the location of the output files as is (the default value is the directory path for the input data file), or you can click Set to specify a folder for storing the output files, a different location for the folder, or both.
9. Optionally, before you process the files, click Save to save the settings that you have specified to a Settings file (.ini file).
 | You can always load this file at a later date and process other data files according to the saved settings in the file. |
10. Click OK.
A message opens the process is completed. If you selected “Determine Automatically” and you did not specify the total tag count, then two mutually exclusive criteria are used to determine when sorting by true tag sequences is complete.
• When the count of reads that contain a sample tag is less than 10% of the count for the previous tag, the tag is not used and barcode sorting is complete.
• After 95% of the sample reads have been parsed by barcode, one additional tag is used for sorting and then sorting is completed.
The names of the separate data files that are produced by the parsing are appended with the following information:
• The tag information as shown (if “Determine Automatically” was selected).
• The sample ID (if a Barcode/Primer file was used).