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  • Introduction
  • Outline
  • Transcript Diversity
  • Current State of Transcript Assembly
  • Difficulties for Resolving Transcripts with Short Reads
  • Iso-Seq™ Method: PacBio® Sequencing for Isoform Analysis
  • Iso-Seq™ Library Preparation
  • Clontech® SMARTer® cDNA synthesis
  • PCR Optimization and Size Selection
  • Iso-Seq™ Library Preparation
  • Size Selection is Necessary for Loading Longer Transcripts
  • BluePippin™ System as an Alternative to Gel Cutting
  • Why Perform a Double BluePippin™ Selection?
  • Iso-Seq™ Library Preparation using BluePippin™ System
  • Goal of Quality Control
  • Identify Full-Length (FL) Reads
  • Expected FL% at Different Size Ranges
  • Validate size selection by plotting FL read lengths
  • Artificial Chimeras (1)
  • Artificial Chimeras (2)
  • Bioinformatics QC Summary
  • Iso-Seq™ Bioinformatics: Complete Overview
  • Why Isoform-level Clustering?
  • MCF-7 Dataset
  • Length Distribution of Final Dataset
  • Number of Isoforms per Loci
  • UCSC browser screenshot of the BRCA1 gene region
  • Unannotated transcript in UCSC genome browser
  • UCSC browser screenshot of the antisense gene pair KIAA0753-MED31
  • Candidate Cancer Fusion Genes
  • Literature-supported Fusion Genes
  • Known cancer fusion gene BCAS4/BCAS3 identified
  • MCF-7 Data Release
  • Rat Heart and Lung Transcriptome
  • Consensus Transcript Length & Accuracy
  • Multiple isoforms observed at a single loci
  • Comparison between Rat Heart and Lung
  • Iso-Seq™ Bioinformatics: Isoform-level Clustering
  • Goal for Iso-Seq™ Protocol
  • Iso-Seq™ Bioinformatics
  • Isoform-level clustering: Background
  • Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory
  • Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data
  • Finding Isoform Clusters
  • Finding isoform clusters through pairwise alignment
  • Defining an isoform hit from an alignment
  • Differentiating true structural differences from errors
  • Full length, non-chimeric reads
  • Reassignment of Reads based on Likelihood
  • Merge Highly Identical Clusters
  • Form New Clusters
  • Iterative Clustering for Error Correction
  • Tricks for speeding up
  • Quiver for Final Consensus Polishing
  • Isoform-level Clustering: Overview
  • Clustering Example
  • Collapsing Redundant Transcripts
  • High-quality, non-redundant, full-length isoforms
  • GitHub Code Repository
  • Iso-Seq™ Summary
  • Iso-Seq™ References
  • Q & A
  • Do you need to optimize the number of cycles?
  • How much is the total RNA input?
  • It looks like you start with sub-reads, why not CCS reads?
  • For the accuracy, what is the sequencing depth requirement?
  • How independent is the bioinformatics pipeline?
  • Possibilities for combining with Illumina data?
  • What is the number of reads needed to detect expression and differential expression?
  • Does the protocol require Hot Start?
  • When you start using reads of insert they are not error corrected by other passes of the same reads?
  • RNA's of different sizes will be loaded into different SMRT cells after size selection?
  • How about using low-input protocol?
  • Would you run ICE before GMAP?
  • Would you recommend using MagBead?
  • What is the minimum number of SMRT cells needed for transcriptome sequencing?
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  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
  • Speaker: Elizabeth Tseng, Senior Bioinformatics Scientist / Pacific Biosciences
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