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Enhance your resequencing and variant detection capabilities

SMRT Analysis provides a rich set of algorithms and applications for sequence alignment, consensus calling, polishing, and variant detection. These tools offer tailored analytical workflows for various scientific applications and provide standardized results. In addition, comprehensive reports are available for visualization and data mining.

SMRT Analysis algorithms for alignment, consensus calling, polishing, and variant detection

BLASR

Basic Local Alignment with Successive Refinement (BLASR) rapidly maps reads to genomes by finding the highest-scoring local alignment or set of local alignments between the read and genome. Optimized for PacBio’s extraordinarily long reads and utilizing rich quality values, BLASR maps reads rapidly and accurately.

Quiver

Quiver finds the maximum likelihood template sequence based on PacBio template reads. PacBio reads are modeled using a conditional random field approach that assigns a probability to a read given a template sequence. In addition to the base sequence of each read, Quiver uses QV values from base calling, allowing for more accurate consensus calls.

LAA

Long Amplicon Analysis (LAA) finds phased consensus sequences from a pooled set of polyploid amplicons that have been sequenced using PacBio’s SMRT technology. The LAA has four main analysis steps: coarse clustering, fine phasing, consensus building, and post-processing. Use this analysis to process reads from the insert sequence of multiple molecules.

Learn more about using PacBio technologies for single-molecule sequencing.

Coarse clustering is used to find groups of reads coming from different amplicons. The reads from each coarse cluster proceed to the phasing step, which separates full-length haplotypes using a Quiver-based phasing algorithm. The haplotypes are then polished using the Quiver algorithm to achieve a high-quality consensus sequence. Finally, in post-processing, spurious consensus sequences and sequences representing PCR artifacts are identified and removed.

CCS

The Circular Consensus Sequencing (CCS) algorithm calculates consensus sequences from multiple subreads — also known as “passes” — around a circularized single DNA molecule (SMRTbell template). CCS uses the Quiver framework to achieve optimal consensus results given the number of passes available. Use this algorithm to process reads from the insert sequence of single molecules and estimate the length of the insert sequence loaded onto a SMRT Cell.

Learn more about using PacBio technologies for single-molecule sequencing.

 

SMRT Analysis applications for alignment, consensus calling, polishing, and variant detection

BAM_Resequencing_Beta.1

This application is used for whole-genome or targeted resequencing workflows. Analysis filters read and map them to a provided reference sequence. It identifies consensus sequence and calls variants using Quiver. BAM_Resequencing_Beta.1 utilizes the BAM file format during analysis, and is significantly faster than RS_resequencing.

RS_Resequencing

This application is used for whole-genome or targeted resequencing workflows. Analysis filters read and map them to a provided reference sequence. It identifies consensus sequence and calls variants using Quiver.

RS_Minor Variant

This application calls minor variants in a heterogeneous data set against a user-provided reference sequence. It features quantitative detection sensitive enough to spot 1% genomic variants in a DNA sample.

RS_Long_Amplicon_Analysis

This application is used to determine phased consensus sequences for pooled amplicon data. It allows for accurate allelic phasing and variant calling in large genomic intervals. The LAA analysis application supports analysis of novel haplotypes in loci of interest, including the medically important HLA region in the human genome. It can pool up to five distinct amplicons. Reads are clustered into high-level groups, then each group is phased and consensus is called using the Quiver algorithm. If the sample is barcoded, analysis can optionally split reads by barcode. Use this analysis to process reads from the insert sequence of multiple molecules.

Learn more about using PacBio technologies for single-molecule sequencing.

RS_ReadsOfInsert

This application analyzes reads from single molecules. It generates a consensus sequence for each molecule and estimates the length of the insert sequence loaded onto a SMRT Cell. If the sample is barcoded, analysis can optionally split reads by barcode. Use this analysis protocol to process reads from the insert sequence of single molecules.

Learn more about using PacBio technologies for single-molecule sequencing.

RS_ReadsOfInsert_Mapping

This application generates consensus sequences from single molecules. It filters reads, maps them to a provided reference sequence, and identifies consensus and haploid variants against this reference.

Use this analysis protocol to process reads from the insert sequence of single molecules.

Learn more about using PacBio technologies for single-molecule sequencing.

Additional resources

  1. Learn More about BLASR
  2. Learn More about Quiver
  3. Quiver FAQ
  4. Variants.gff File Format Specifications
  5. Minor Variants and Phasing Analysis

 

Contact us to learn more about how this innovative software can support your research efforts.

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