Table 2 Steps in performing a sequence walk

1.

Open the file containing the initial target sequence (usually one or more melded reads).

2.

Select direction of walk (it is operationally simpler to walk 3’ to 5’ first because this does not require later shifting the matching index of any previously matched reads).

3.

Select the target length, minimum base overlap (typically >30), maximum permissible errors per match (typically 0–2), minimum number of bases to meld a ragged end (typically >4), maximum number of reads to find per step (typically 50) and maximum number of reads to add per step (if less than the maximum to be found).

4.

Select the base orientations to test (depends on the data available - forward and reverse complement for an Illumina paired-end set).

5.

Click ‘Walk’. The program will ask for one or two data files to search, and then proceed.

6.

The program will finish walking when the data is exhausted (no more matching reads found) or the operator clicks ‘Stop’. At this point, any duplicate sequences can be removed (menu) and the data files can be searched for the missing member of any incomplete pair (menu). Any extra paired reads found can be matched to the main sequence (menu).

At the end of a 3’ to 5’ walk the dialog will indicate the size of the origin shift (in bases). The matching sequences from any previous walk can then be shifted by that amount (menu). All matching reads (from both walks and any additional paired ends) can be combined into one set (menu). The combined set can then be melded to give the complete discovered sequence (menu).