RS_Resequencing_Barcode Protocol

Use this protocol to map length and quality-filtered reads against a reference sequence, then to identify consensus and variant sequences using the Quiver algorithm.

• Used for whole-genome or targeted resequencing where your samples are barcoded.

• Filters reads, maps them to a provided reference sequence, and then calls a consensus sequence.

Filtering Parameters (SFilter v1)

• Minimum Subread Length: Subreads shorter than this value (in base pairs) are filtered out and excluded from analysis.
• Minimum Polymerase Read Quality: Polymerase reads with lower quality than this value are filtered out and excluded from analysis.
• Minimum Polymerase Read Length: Polymerase reads shorter than this value (in base pairs) are filtered out and excluded from analysis.

Mapping Parameters (BLASR Barcode 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.

Barcode Parameters (Barcode Module v1)

• My Library has DNA Barcodes That Are:
• Paired: A pair of barcode sequences that always occur together, and are different on each end.
• Symmetric: A single barcode sequence that occurs at both ends of the insert sequence.
• Barcode FASTA FIle: Path to the FASTA file containing the barcodes to use.
• Minimum Barcode Score: The minimum score for calling a barcode. This must be between 0 and 2 times the length of the barcode.