Use CasesBarcode Classification

---

Torrent Suite Software space on Ion Community

Use Cases TOC

Custom Barcode Design

Cautions

Custom barcode design involves certain technical challenges:

• Calculation of the your barcodes' hamming distances in flow space

• Adjustment of basecaller parameters to match your barcodes' distances

Custom barcode design is for advanced users only and only if you have a compelling need for a custom barcode set.

If are considering creating your own custom barcode set, we recommend that you first contact your FBS.

IMPORTANT : The default Basecaller parameter settings are optimized for the IonXpress barcode set. The use of a different barcode set, especially a custom barcode set, requires custom Basecaller parameter settings.

Barcode overview

The Torrent Suite Software supports barcoded runs, in which multiple barcoded samples are processed on the ION Chip during an Ion sequencing run. A barcode run typically involves sample-prep with an Ion barcode adapter kit (or compatible kit) such that two or more barcode adapters are present in a run. The user selects the barcode set in the run Planning tab of the Torrent Browser. This barcode set information is used during analysis to separate out reads by barcode, remove the barcode and adapters from the read, and output reads by barcode into separate BAM files.Reads are aligned against the reference genome, and results stored in BAM and BAM index (BAI) files for each barcode. Reads that can not be classified as being one of the barcodes in the designated set are grouped into a "no-match'" group, and alignment against the reference also performed on theno-matchgroup.

Alignment metrics for each barcode are available in the Output Files section of the analysis run report.The run report shows Q20 performance metrics for all barcodes in the run, providing a quick glance at the high-level quality of each barcode.The barcode section in the run report also shows the following metrics for each barcode in the run:

• The number of bases

• The number of bases at Q20 (or better) accuracy

• The number of mapped reads

• The mean read length

• A read length histogram

The Torrent Suite Software includes barcode sets forthe latest available barcode kits. These barcode sets are selected in the run Planning tab.Advanced users optionally can add additional barcode sets in the References section of the Torrent Browser admin tab, either by uploading a CSV file of all barcodes or by manually adding each barcode.

Barcode set design considerations

Barcode sets are designed to efficiently separate reads from each other in the presence of errors. Ion Torrent sequencing technology produces raw data in flow space. These reads are best described as having a homopolymer run of length 0, 1, 2, etc., ... in flow 1, 2, 3, etc. Because of this characteristic, the most typical error patterns involve either over- or under-estimation of a homopolymer signal in a flow. The most effective barcodes designs for Ion Torrent technology are those with distinctive flow-space representations.

Hamming distance

One way of describing the separation of two sequences in flow space is by the hamming distance between them for relevant flows. Hamming distance is the number of flows in which the expected homopolymer length is different between the two sequences. For example, if two barcodes differ in 5 flows in flows 9-22, those two barcodes have hamming distance 5.

Hamming distance corresponds naturally to the ability to detect and correct errors. When two sequences have hamming distance 5, 2 errors can occur on one of the sequences and that sequence is still 3 errors away from the other sequence. Sequences separated by hamming distance 5 can tolerate 2 errors and still be classified correctly.

Ternary encoding

One side effect of operating in flow space is that barcodes are not limited to binary sequences. For example, each flow can correspond to 0, 1, or 2 bases in a ternary encoding scheme. This scheme allows for a greater number of codewords occupying the same number of flows. However, a flowspace representation must correspond to a legitimate sequence that yields these flow-space values.For example, we cannot have a flow of T, C, T with values 1, 0, 1.For a flow of T, C, T, bothT bases are consumed in the first flow, andthe sequencing reaction yields the incorrect values 2, 0, ?.

Ion Torrent b arcode design

We designs Ion Torrent barcode sets to provide at least 1-error correction (hamming distance 3) in flow space for a large set of barcodes, and 2-error correction (hamming distance 5) for a usefully sized subset of such codes.This goal is accomplished by taking the ternary hamming code on 13 characters and assigning codewords to flows 9-22 to generate flow sequences (flows 1-8 are used for the library key and are not considered here). These flow sequences then have hamming distance 3 and are 1-error correcting.The codewords are further reduced by the constraint of requiring that they correspond to legitimate flow sequences.We also apply the constraint that the flow sequences must correspond to base sequences that are 9 to 11 bases in length.Finally, within the set that satisfies all these constraints, a subset is chosen (by greedy aggregation) such that any pair of flow sequences has hamming distance 5.

To insulate these sequences from the target sequences, a ligation adaptor CGAT is added. The ligation adaptor performs two functions. First, the C in flow 22 provides a synchronized flow that both marks the end of the barcodes and ensures that barcodes ending with "0" do not have sequence overwrite those flows. Secondly, this adapter mitigates any sequence-specific biases caused by the differing barcode sequences.

We provide a tool that classifies barcode reads by finding the flow-space representation of the read and comparing it to the flow-space representation of the barcodes. Classification standardly occurs after the last flow of the key (G), and continues to the end of the barcode sequence provided in flowspace. Ion Torrent barcode sets are designed to be synchronous so thatthey all are classified using the same set of flows.

For flow space classification of custom barcodes, the barcodes should be designed to be compatible with the flow order, be synchronized at a final flow, and be well separated. However, the Torrent Suite Software attempts to classify any reasonable set of sequences that are separated in flow space.Many standard software packages also classify usefully in sequence space, and have been found to work well with Ion Torrent data.

Related information

Manage DNA Barcodes and DNA Barcode Sets : Explains the mechanics of using Torrent Browser to add a new barcode set.

Other BaseCaller and barcode classification pages:

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

---