CSC

Mutation Scanner

Description

This module compares each input sequence chromatagram against a reference chromatogram (or trace) to detect mutations. The reference traces are specified in the see section Reference Traces and Reference Sequences. Using this method it is possible to detect both base-change mutations and heterozygous mutations. It works by aligning the reference trace with the input trace and then examining the peak pairs for each individual base separately. It does not use basecalls as these are prone to error and their use generates too many false positives. After normalisation, the amplitude ratios of peak pairs which are abnormal are analysed more closely. For heterozygotes, a drop in peak height with respect to the reference of about 50% is expected. The final set of candidate mutations are validated against a difference trace to ensure it contains a double peak at that location, thus confirming the mutation to be real. After chromatagram analysis has been completed, mutation tags are written back to the Experiment File as HETE and MUTA tags.

Option: Adaptive Noise Floor

Traces are very noisy difficult to process signals. To find valid peaks in a trace an adaptive noise threshold based on envelope height is used to eliminate all low level noise from consideration. The effect of this parameter can be seen in the trace below. By default this parameter is set to 25% of envelope peak height. If set lower, too much noise is picked up; if set higher, low level mutations may be missed.

Option: Upper and Lower Peak Drop Thresholds

For heterozygote mutations, the peak height of the mutant drops by 50% with respect to the normalised reference trace as shown in the trace below. For accurate detection, we use this information to validate potential mutations. Due to overzealous preprocessing done by sequencing machine software, the peak height drops are often not 50%, but typically hover between 20% and 70% of reference peak height. Any potential heterozygote whose peak height drop with respect to the normalised reference trace that lies within this range is considered to be a real mutation.

Option: Peak Alignment Search Window Size

In an ideal world, heterozygote peaks in a trace would be perfectly aligned on top of each other. In practice however, they can often be skewed due to gel chemistry problems or inaccurate mobility correction as shown in the trace below. When mutscan looks for peak pairs, it allows for this skew by looking either side of the current position for nearby peaks. This parameter is the distance mutscan looks in bases around each candidate position.

Option: Heterozygote SNR Threshold

For a normal trace containing normal bases, the signal-to-noise ratio (SNR) is the ratio of the highest base peak to the second highest trace level. Mutscan computes this value in decibels (dB) as 20*log10(S/N). For normal bases, this usually in the region of 20-30dB or higher. However, for heterozygotes, the SNR as defined by this measure degrades significantly to around 2-5dB. This is the mechanism mutscan uses to accurately determine the mutation tag type. If the candidate mutation's SNR is equal to or below this threshold, mutscan designates it to be heterozygous, otherwise it's considered to be a normal base-change mutation.

Option: Trace Alignment Failure Threshold

Mutscan works by aligning a mutant trace against a reference trace and comparing the peaks. However, if the traces are too different, the alignment may fail and as a consequence, large numbers of false positive mutation tags are generated. Typically, within each trace there are only one or two mutations, so if we find 15 mutations, then we can confidently predict that things have gone badly wrong! This parameter sets a threshold, beyond which an alignment failure error message is printed, rather than outputting large numbers of invalid mutation tags.

Option: Complement Bases on Reverse Strand Tags

After mutation detection and after readings have been assembled into a GAP4 database, GAP4 displays both forward and reverse readings in a single direction in the contig editor. This makes it much easier to compare sequences and traces in both directions simultaneously. When the corresponding traces are displayed, any reverse strand traces are complemented automatically such that the bases are interchanged. In this case, the original mutation tag generated by mutscan will then be of the wrong sense. If checked, this option complements the tag base labels to match the complemented trace displayed by GAP4.