Contents
What is needed in the base quality score recalibration process?
Amount of data. A critical determinant of the quality of the recalibration is the number of observed bases and mismatches in each bin. This procedure will not work well on a small number of aligned reads. We usually expect to see more than 100M bases per read group; as a rule of thumb, larger numbers will work better.
Why base quality score recalibration?
The purpose of base quality score recalibration is to correct for biases in the quality scores assigned by the sequencer. The idea is to compute new base quality scores for each read by applying an error model derived from the full data set.
What is Mutect2?
Given a matched normal, Mutect2 is designed to call somatic variants only. The tool includes logic to skip emitting variants that are clearly present in the germline based on provided evidence, e.g. in the matched normal. This is done at an early stage to avoid spending computational resources on germline events.
Why does base quality go down towards the end of the read?
The main reason for the decreasing sequence quality is the so-called phasing. Phasing means that the blocker of a nucleotide is not correctly removed after signal detection.
What is RMS mapping quality?
Root Mean Square of the mapping quality of reads across all samples. This annotation provides an estimation of the overall mapping quality of reads supporting a variant call, averaged over all samples in a cohort.
What is genotype quality?
The Genotype Quality represents the Phred-scaled confidence that the genotype assignment (GT) is correct, derived from the genotype PLs. Specifically, the GQ is the difference between the PL of the second most likely genotype, and the PL of the most likely genotype.
What is a panel of normals?
The Panel of Normals approach uses a set of matched normal samples to determine the baseline level from which to call CNV events. These matched normal samples should be derived from the same library prep and sequencing workflow that was used for the case sample.
What is somatic calling?
The somatic variant caller was designed for instances where a single cancer sample has been sequenced; it is not intended for analyzing tumor/ normal pairs. For SNP calling, the somatic variant caller considers each position in the reference genome separately, starting with the bases of the aligned reads.
What’s the process for calibrating a quality score?
Here we present an eight-step process for calibrating quality scores that combines calibration sessions with gathering data and looking for deviations.
Where can I use base quality score recalibration?
This procedure can be applied to BAM files containing data from any sequencing platform that outputs base quality scores on the expected scale. We have run it ourselves on data from several generations of Illumina, SOLiD, 454, Complete Genomics, and Pacific Biosciences sequencers. That sounds great! How does it work?
What are the different types of tool calibration?
There are 3 types of standards in your tool calibration and control system: Primary Standards: Those units or instruments of highest quality and stability which are periodically calibrated by the National Institute of Standards and Technology (NIST) or by other contract calibration services which have direct traceability to NIST.
How much accuracy do you need for tool calibration?
Select accuracy data points from the low, mid and high range of the equipment. The known standard must have a tighter accuracy then the instrument. For calibration of dimensional tools, the known standard should have a 10 to 1 accuracy of the instrument.