Translocation Analysis

  • Updated

Translocations are a type of rearrangement in which sections of DNA are exchanged within or across chromosomes. Interchromosomal translocations occur across nonhomologous chromosomes and may include reciprocal translocations (where parts of two chromosomes switch places) or nonreciprocal translocations (where a piece of one chromosome breaks off and attaches to a different chromosome in a 1-way transfer, see figure below).

 

Reciprocal & nonreciprocal translocations

 

 

Translocations are a possible undesired editing outcome of genome editing, especially when two double-strand breaks are generated using a gene editing tool. Because knockout generally involves double-strand break generation and base editing does not, translocation analysis is included only in the Knockout Report (single-sample and multi-sample), but not in the Base Editing Report. 

 

The Tapestri Genome Editing Pipeline detects translocations across genome editing targets (i.e., between on-targets, between predicted off-targets, and between on and off-targets). Deletions ≳ 2,000 bp may also be detected between two known target sites on the same chromosome. 

 

The translocation detection module checks for chimeric reads in each cell (note: this happens after "Step 3: Genome alignment" step and before "Step 4: Genotype calling" step based on the steps listed in the Algorithms Overview support article).

Chimeric reads begin with the primer of one amplicon and end with the primer of another. These amplicons must be from different chromosomes or at least 2,000 bp apart on the same chromosome. If chimeric reads are found between amplicons on the same chromosome, it could indicate a large deletion between them.

The pipeline uses two filters to minimize false positives when detecting translocation events:

  1. At least five reads must support a translocation event in a cell.
  2. The ratio of chimeric to normal reads must exceed 0.15 for a translocation event in a cell.

The chimeric-to-normal read ratio is calculated by dividing the number of chimeric reads (those spanning two amplicons) by the average number of properly paired reads for those amplicons in the same cell. This method assumes that when a translocation occurs in just one of the DNA copies in a cell, both chimeric and normal reads will be present. Since these reads come from the same DNA copy, their quantities should be similar. If only chimeric reads are found in a cell, this ratio-based filter is not applied.

Panel design considerations

The specific translocations that can be detected are determined during the custom panel design process. No additional panel files are needed for translocation detection. However, the types of translocation products detected are dependent on primer design. Mission Bio’s White Glove team is available to assist in panel design so that desired types of translocations can be measured and to assist in complex cases where both targets are on the same chromosome. To learn more about our White Glove service, and panel requirements please contact our support team (support@missionbio.com). Additional panel recommendations can be found here.

For reciprocal translocations — in which parts of two different chromosomes swap places — four resulting chromosome products are possible: two monocentric (one centromere), one acentric (no centromere), and one dicentric (two centromeres, see figure below). 

However, due to the way the primers are designed, only two of these four types can be detected using this method. This is because only certain primer combinations include both necessary sequencing adapters, allowing for successful library preparation for sequencing.

The potential products of reciprocal translocations are depicted below. Please note that a single Tapestri Genome Editing assay can detect either two monocentric translocations or one acentric and dicentric translocation in a single assay.

 

Potential translocation products between two p-arm loci

Note: p1 and p2 demarcate each side of a double-strand break on the p-arm.

 

Potential translocation products between two q-arm loci

Note: q1 and q2 demarcate each side of a double-strand break on the q-arm.

 

Potential translocation products between one p-arm and one q-arm loci

Note: p1 /p2 and q1/q2 demarcate each side of a double-strand break on the p-arm and q-arm, respectively.

 

For nonreciprocal translocations — in which part of one chromosome transfers to another in a one-way fashion — two resulting chromosome products are possible: the chromosome that provided the genetic material and the chromosome that received the genetic material. The ability to measure these translocations also depends on the primer design of the panel. A nonreciprocal translocation from the p arm of chromosome 2 to 1 is shown below for illustrative purposes. 

Note: p1 and p2 demarcate each side of a double-strand break on the p-arm.

 

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