## Using quality scores when merging

Quality scores come into play in two different ways when merging overlapping pairs.

First, if there is a conflict between the reads in a pair (i.e. a mismatch or gap in the alignment), quality scores are used to determine which base the merged read should have at a given position. The base with the highest quality score will be the one used. In the case of gaps, the average of the quality scores of the two surrounding bases will be used. In the case that two conflicting bases have the same quality or both reads have no quality scores, an [IUPAC ambiguity code](see IUPAC codes for nucleotides) representing these bases will be inserted.

Second, the quality scores of the merged read reflect the quality scores of the input reads.

We assume independence of errors in calculating the new quality score for a merged position and carry out the following calculations:

• When the two reads agree at a position, the two quality scores are summed to form the quality score of the base in the new read. The score is capped at the maximum value on the quality score scale which is 64. Phred scores are log scores, so their sums represent the multiplication of the original error probabilities.
• If the two bases disagree at a position, the quality score of the base in the new read is determined by subtracting the lowest score from the highest score of the input reads. Similar to the addition of scores when bases are the same, this adjusts the error probability to reflect a decreased certainty that the base reported at that position is correct.
Thus, if two bases at a given position of an overlapping region are different, and each of those bases was originally given a high phred score, the score assigned to the merged base will be very low. This reflects the fact that the base at this position is unreliable.

If a base at a given position in one read of an overlapping region has a very low quality score and the base at that position in the other read has a high score, it is likely that the base with the high quality score is correct. The adjusted quality score for this position in the merged read would reflect that there is less certainty in the base at that position than before. However, such a position would still be assigned quite a high quality, as the base call is still likely to be correct.