RS_Minor_Variant  Protocol (Beta)

Use this protocol to call minor variants in a heterogeneous data set against a user-provided reference sequence.

• Features quantitative detection to 1% genomic variants in a DNA sample.
• Uses a more sophisticated model tuned to PacBio reads.

• Note: This protocol replaces the RS_Minor_and_Compound_Variants protocol in previous releases.

The protocol includes two main steps:

1. Realignment/Data Acquisition: Unambiguously re-map query sequences to the reference in an unbiased manner, avoiding the reference bias problem of straight alignment.

2. Variant Detection: Call variants above sequencing noise using the LoFreq algorithm.

Data Prep Parameters (Reads Of Insert)

• Minimum Full Passes: The minimum number of full-length passes over the insert DNA for the read to be emitted.
• Minimum Predicted Accuracy: The minimum predicted accuracy (in %) of the reads of insert emitted.
• Minimum Length of Reads of Insert (In Bases): Reads of insert shorter than this value (in base pairs) are filtered out and excluded from analysis.
• Maximum Length of Reads of Insert  (In Bases): Reads of insert longer than this value (in base pairs) are filtered out and excluded from analysis.

Mapping Parameters (BLASR RoI v1)

• Maximum Divergence (%): The maximum allowed divergence of a read from the reference sequence.
• Minimum Anchor Size: The minimum size of the read (in base pairs) that must match against the reference sequence.
• Write Output as a BAM File: Specify whether or not to output a BAM representation of the cmp.h5 file.
• Write BED Coverage File: Specify whether or not to output a BED representation of the depth of coverage summary.
• Place Repeats Randomly: Specify that if BLASR maps a read to more than one location with equal probability, then it randomly selects which location it chooses as the best location. If not set, BLASR defaults to the first on the list of matches.

Variants Parameters (MinorVariants v1)

• Minimum Subread Length: Subreads shorter than this value (in base pairs) are filtered out and excluded from analysis.
• Minimum Site Coverage: The minimum required site coverage to score variants at that site.
• Minimum Subread Alignment Accuracy: The minimum alignment accuracy of a subread.
• Maximum P-value: The maximum probability of obtaining a test statistic at least as extreme as was observed for this variant; a threshold for reporting variants. Specifies whether something is sufficiently different from our null model to warrant further investigation. Lower p-values are more significant; variants with large p-values are probably a result of machine error.
• Frequency Confidence Interval: The confidence interval to report about the frequency. (Each variant occurs at a given frequency within a sample. Example: For a value of 0.95, we have a roughly 95% confidence that the frequency is between the reported interval.)
• Call Amino Acid Variants: Specify whether or not to report the name of the variant in amino-acid space. (Example: E662K for a reference glutamic acid (E) at position 662 mutated into a lysine (K)). This can simplify analysis.  
  Note: You must provide an in-frame reference translatable end-to-end with no more than a terminal stop codon.