IsoPhase and Cogent

Elizabeth Tseng, Principal Scientist, PacBio

IsoPhase: Isoform-Level Phasing

ISOFORM-LEVEL PHASING USING ISO-SEQ READS

ISOPHASE: ISOFORM-LEVEL PHASING

MATERNAL IMPRINTING IN MAIZE

PARENTAL-SPECIFIC ISOFORM EXPRESSION IN MAIZE

PHASING BEYOND DIPLOID – A CHALLENGE

TISSUE-SPECIFIC ALLELIC EXPRESSION IN CATTLE

ISOPHASE ON GITHUB (PART OF CUPCAKE)

Click here for haplotyping using Iso Seq data

Cogent: Coding Genome Reconstruction Without A Reference Genome

NO GENOME? NO PROBLEM

COGENT workflow

• Using only Iso-Seq data to find gene families and reconstruct a fake “genome”

Click here

Use COGENT results to…

• Evaluate genome assemblies
  • Pig Iso-Seq Cogent rescued 5 missing genes for the new pig assembly
• Visualize alternative splicing
  • You can still see skipped exons!

COGENT: WHY?

Not every species has a high-quality reference genome

• If genome is poor, genome annotation (ab initio prediction & mapping) will suffer

Iso-Seq bioinformatics does not require a genome

• There is often enough information in Iso-Seq transcripts itself to identify gene families and the “coding” regions of the genome

COGENT: HOW

COGENT: GENE FAMILY FINDING

GENE FAMILY PARTITIONING OF 9 HUMAN GENES

COGENT: GENOME RECONSTRUCTION

Full Cogent algorithm here

COGENT CASE STUDY: PIG GENOME EVALUATION

COGENT CASE STUDY: MAKING A “FAKE CODING” GENOME

Click here for the tutorial

Genome-based View

• All introns are visible

Cogent-based View

• Introns that are never transcribed are not visible

WHY COGENT MAY SOMETIMES OUTPUT >1 CONTIGS

• Lack of connectivity information between exon 1 and 2 based solely on transcripts
• Cogent outputs two contigs, one with exon 1 - 3, one with exon 2 – 3
• Mapping back to genome shows that the reconstruction is correct
• This is a case where genome information can be used to order exon 1 and 2

Coming soon: Special Cogent parameter to make “best guess” for ambiguity

The pdf to this documentation can be found here

Special Iso-Seq topic: Phasing and Cogent (hands on)

The Iso Seq Bioinformatics Tutorial we are using is here. See ‘3. Cogent’ and answer the corresponding practice questions!