Secondary Analysis Protocols Provided by Pacific Biosciences

Following are descriptions of the secondary analysis protocols provided by Pacific Biosciences. (Click the links for more detailed information.)

BAM_Resequencing_Beta (Beta)

• 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.
• This is an experimental version of the RS_Resequencing protocol which uses BAM rather than cmp.h5 as the output file format.
• This protocol is faster than RS_Resequencing for large jobs, but is not yet guaranteed to produce identical results.

RS_AHA_Scaffolding

• Use this protocol to build hybrid assemblies up to 200 Mb in size with PacBio reads.

RS_BridgeMapper

• Use this protocol to troubleshoot de novo assemblies, variants, indels, and so on.

RS_HGAP_Assembly.2

• Use this protocol to perform de novo assembly using a single PacBio library prep. (The protocol is optimized for quality.)

• Note: This protocol replaces the RS_HGAP_Assembly.1 protocol in previous releases.

RS_HGAP_Assembly.3

• Use this protocol to perform de novo assembly using a single PacBio library prep. (The protocol is optimized for speed.)

RS_IsoSeq

• Use this protocol to classify, cluster, and map cDNA sequences.

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

RS_Long_Amplicon_Analysis

• Use this protocol to determine phased consensus sequences for pooled amplicon data.

RS_Minor_Variant (Beta)

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

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

RS_Modification_Detection

• Use this protocol to identify common bacterial base modifications, including 6-mA, 4-mC, and optionally TET-converted 5-mC.

RS_Modification_and_Motif_Analysis

• Use this protocol to identify common bacterial base modifications (6-mA, 4-mC, and optionally TET-converted 5-mC), and then analyze the methyltransferase recognition motifs.

RS_PreAssembler

• Use this protocol to build a set of highly accurate long reads for use in de novo assembly.

RS_ReadsOfInsert

• 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.

• Replaces the Circular Consensus Sequencing (CCS) protocol, which has been moved off the primary analysis instrument as of v2.1

RS_ReadsOfInsert_Mapping

• Use this protocol to generate consensus sequences from single molecules.
• Note: This protocol replaces the RS_Resequencing_ReadsOfInsert protocol in previous releases.

RS_Resequencing

• 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.

RS_Resequencing_Barcode

• 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.
• Use this protocol if your samples are barcoded.

RS_Site_Acceptance_Test

• Use this protocol to generate a report displaying site acceptance test metrics.

RS_Subreads

• Use this protocol to filter reads based on the minimum read length and read quality you specified.
• Note: This protocol replaces the RS_Filter_Only protocol in previous releases.