Mapping paired reads

The CLC Genomics Workbench offers as the default choice to automatically calculate the distance between the pairs. If this is selected, the distance is estimated in the following way:

  1. A sample of 200,000 reads is extracted randomly from the full data set and mapped against the reference using a very wide distance interval.
  2. The distribution of distances between the paired reads is analyzed using a method that investigates the shape of the distribution and finds the boundaries of the peak.
  3. The full sample is mapped using this distance interval.
  4. The history (Image history_16_n_p) of the result records the distance interval used.
The above procedure will be run for each sequence list used as input, assuming that they do not necessarily share the same library preparation and could have different distributions of paired distances. Figure 30.7 shows an example of the distribution of intervals with and without automatic pair distance interval estimation.

Image before_and_after_pair_estimation
Figure 30.7: To the left: mapping with a narrower distance interval estimated by the workbench. To the right: mapping with a large paired distance interval (note the large right tail of the distribution).

Sometimes the automatic estimation of the distance between the pairs may return a warning "Few reads mapped as pairs so pair distance might not be accurate". This message indicates that fewer than 10.000 paired reads were available for estimation of the paired distance, hence the estimated distance may not be accurate. If in doubt, you may want to disable the option to automatically estimate paired distances and instead manually specify minimum and maximum distances between pairs on the input sequence list.

If the automatic detection of paired distances is not checked, the mapper will use the information about minimum and maximum distance recorded on the input sequence lists (see General notes on handling paired data).

If a large portion of pairs are flagged 'Broken' we recommend the following:

  1. Inspect the Detailed mapping report to deduce a distance setting interval - and compare this to the estimated distance used by the mapper (found in the mapping history).
  2. Open the paired reads list and set a broad paired distance in the Elements tab. Then run a new mapping with the 'auto-detect...' OFF. Make sure to have a report produced. Open this report and look at the Paired Distance Distribution graph. This will tell you the distances that your pairs did map with. Use this information to narrow down the distance setting and perhaps run a third mapping using this.
  3. Another cause of excessive amounts of broken pairs is misspecification of the read pair orientation. This can be changed in the Elements tab of the paired reads list prior to running a mapping.

See Detailed mapping report for further information.

When a paired distance interval is set, the following approach is used for determining the placement of read pairs: