---

Torrent Suite Software space on Ion Community

User Interface Guide TOC

BaseCaller Parameters

This page describes BaseCaller parameters that are available when you reanalyze a completed run.

Changes in the 4.4 release

Barcode filtering

Barcode classification

Changes in the 4.2 release

Barcode classification method

The barcode classification method that is used in the Torrent Suite Software analysis pipeline is changed beginning with the 4.2 release. Barcodes now are classified based on signal information rather than on base call information, as was done in 4.0 and previous releases. S ignal calls are a more direct measurement of the read and are the most precise and most accurate source of information. The 4.2 barcode classification method considers the distance between the measured signal and the predicted barcode signal. The 4.0 method considered the number of flow alignment errors in base space.

Advantages of the 4.2 classification method:

• Because this method uses a barcode separation threshold that is based on signal information, it provides increased confidence in the identification and rejection of reads. Using signal information, the BaseCaller can detect problematic reads that in base space appear to perfectly match a barcode.
• The consistency and uniformity of barcode classification is improved. The 4.0 classification method constantly adjusted the classification threshold. In 4.2, the threshold is a defined percentage of the most frequently-occurring barcode (1%, by default).

BaseCaller parameters

The settings of BaseCaller parameters control barcode classification, and these parameters have also changed in the 4.2 release:

• --barcode-mode 2 is the default in 4.2. This setting causes the 4.2 barcode classification behavior.
• The parameter --barcode-cutoff , has a different meaning than in 4.0. In 4.2, --barcode-cutoff sets the maximum allowed squared distance ( between the measured signal and the predicted barcode signal) .

These new parameters are added in the 4.2 release:

• --barcode-separation is minimum separation between the best and second best barcode choice to assign a read to a barcode .
• --barcode-postpone , when set to the 4.2 default of 1, postpones most barcode filtering until after an Ion Proton run's 96 blocks are merged into one. (Some low level pre-filtering still happens at the block level to reduce the number of files that need to be transferred and merged.) Final b arcode filtering is done on the chip's full information as a whole, not on a block-by-block basis.

Note: As always, the 4.2 defaults for these parameters are optimized for the barcode set IonXpress.

Barcode classification for Ion Proton analyses

For Ion Proton sequencing runs, the chip is logically divided into 96 blocks. For signal processing and base calling stages of the analysis pipeline, the data from each block is analyzed independently from other blocks and also from the chip's data as a whole.

4.0

In the 4.0 release, barcode classification and also barcode filtering are done independently in each block. A barcode is reported in the run report if it passes barcode filters in any block. With this approach, noise in a single block can cause an unused barcode to erroneously appear in the run report. Noise could be from various issues such as a loading problem in a small area of the chip or a small bubble in the flow. The edges of bubbles are especially associated with error spikes that cause barcode classification errors in the 4.0 release a single noisy block can cause phantom barcodes to be reported (erroneously) as present in the analysis.

Also in the 4.0 release, reads that fail barcode filtering are filtered out and are not reported in the BAM file or in other files. These reads are silently rejected and cannot be inspected for troubleshooting or other reasons.

4.2

In the 4.2 release, barcode filtering is performed considering all the chip's data as a whole. B arcode filtering is postponed until after the data from all 96 blocks of the chip are merged. We see that this approach avoids many of the phantom barcode issues that are seen in 4.0 due to noise in one of the 96 blocks.

To approximate the 4.0 classification behavior in the 4.2 release

Use the following parameter settings to have the 4.2 release approximate the 4.0 barcode classification behavior:

• --barcode-mode 1
• --barcode-cutoff 2

Due to changes in the behavior of base calibrationand also in the internal quality tables, the 4.2 release cannot exactly reproduce the 4.0 barcode classification behavior.

Basic parameters

This table lists the more common BaseCaller parameters.

The cutoff setting

Notes about the --barcode-cutoff parameter with --barcode-mode 1:

• 0 is the most restrictive setting. --barcode-cutoff 0 allows only reads that perfectly match a barcode in base space.
  
  
  
  
• The setting 0 works with any barcode set (both Ion Torrent sets and custom barcode sets).
  
  
  
  
• Do not set --barcode-cutoff greater than 2 with the IonXpress barcode set. Values greater than 2 relax the classification rules and allow incorrect barcode assignments.

A rule of thumb for the maximum --barcode-cutoff setting is based on the minimum distance of the barcode set in flow space:

T he minimum distance f or the IonXpress barcode set is 5. Then the maximum recommended value for --barcode-cutoff is 2 for analyses that use the IonXpress barcode set. (See Custom Barcode Design for more information about minimum distances within a barcode set.)

The separation setting

Notes about the --barcode-separation parameter:

• Larger values (close to the minimum distance of the code) require more strict matching of the predicted signal for a read to be assigned to a barcode.

• Smaller values (for example, 0.2 and below) allow barcode assignment with an expanded tolerance for errors. For example in the extreme case of separation=0, the measured signal may be right in between two predicted barcode signals.

• If --barcode-separation is set at or above the minimum distance of the barcodes in flow space, no reads at all are assigned to a barcode.
• If --barcode-separation is set close to the minimum distance of the barcodes in flow space, very few reads are assigned to a barcode.
• If --barcode-separation is too small, the risk of cross contamination increases. More ambiguous reads are forced into a barcode assignment (with a higher rate of error in these assignments).

A rule of thumb for a good --barcode-separation setting is one half of the minimum distance of the barcode set in flow space:

Other public parameters

This table lists the public BaseCaller parameters that are available for you to modify. However, please note that the defaults for these parameters are optimized for most scenarios and in most cases the default settings are recommended.

BaseCaller filters

The BaseCaller module and its parameter settings control these types of filtering:

• Keypass
• Quality trimming
• Adapter trimming

(For a conceptual overview of the BacseCaller's trimming, see Overview of BaseCaller and Barcode Classification . For a detailed discussion, see Technical Note - Filtering and Trimming .)

Examples of BaseCaller parameters usage

With these examples:

• Do not remove the string "BaseCaller" from the Basecaller Args field.
• Do not change BaseCaller parameters other than those listed in the basic table or the public table (unless specifically directed to do so by Ion).

Turn off all filtering and trimming

Use the parameter --disable-all-filters on to turn off all filtering. Here is an example Basecaller Args field:

Note: Your analysis most probably contains other parameters in the Basecaller Args field. Do not remove or modify the other parameters.

The --disable-all-filters on setting overrides other filter settings.

Turn off keypass filtering

Use the parameter --keypass-filter off to turn off keypass filtering. Here is an example Basecaller Args field:

Turn off quality filtering

Use the parameter --trim-qual-cutoff 100 to turn off quality filtering. Here is an example Basecaller Args field:

Note: Your analysis most probably contains other parameters in the Basecaller Args field. Do not remove or modify the other parameters.

Turn off adapter filtering

Use the parameter -- trim-adapter-cutoff 0 to turn off adapter filtering. Here is an example Basecaller Args field:

Note: Your analysis most probably contains other parameters in the Basecaller Args field. Do not remove or modify the other parameters.

Assign more reads to barcodes

To assign more reads to barcodes, adjust barcode classification settings towards less stringent settings:

• Increase the -- barcode-cutoff setting
• Decrease the -- barcode-separation setting.

Do not filter out any barcodes

Use the --barcode-filter=0.0 with --barcode-filter -minreads 0.0 to show all barcode read groups where at least one read is classified as belonging to this barcode group.

Other BaseCaller and barcode classification pages

Other resources in Torrent Suite Software user documentation:

• BaseCaller Parameters (including changes in the 4.2 release)
• Troubleshooting Barcode Classification Issues
• Custom Barcode Design
• Filtering and Trimming Tech Note

---