RS_ReadsOfInsert Protocol

Use this protocol to process reads from the insert sequence of single molecules and estimate the length of the insert sequence loaded onto a SMRT Cell.

• Generates reads from the insert sequence of single molecules, optionally splitting by barcode.

• Replaces the Circular Consensus Sequencing (CCS) protocol, which has been moved off the primary analysis instrument as of v2.1.0.
• To obtain the closest approximation of CCS as it existed on-instrument, specify Minimum Full Passes = 2 and Minimum Predicted Accuracy = 0.9 in the Reads of Insert protocol dialog box.

Filtering 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.

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.