AGBT2017 Reference Workshop: Schneider

New technologies, new management challenges for reference assemblies

Valerie Schneider, Ph.D.@dnadiver

NCBI13 February 2017

https://genomereference.org

Twitter: @GenomeRef

Announcements: [email protected]

• Reference assembly management

• Challenges of changing technologies, new resources

• De novo assembly assessment

Evolution of Human Reference Assembly Management

Why do we need data managementand assembly infrastructure?

Reference Assembly Management

Reference Assembly Management

Sanger-seq’d, clone-based assembly BAC insertBAC vector

Shotgun sequence clone

Assemble

GAPS

Finish

Minimal Tiling Path

Define switch points for adjacent clones (haploid mosaic)

Most contiguousHighest sequence quality

Ordering the Path

Fingerprint maps

Genetic linkage maps

Radiation hybrid maps

Component-Level Quality

Shotgun sequence clone

Assemble

GAPS

Finish

Finished: Error rate <10-4

Additional Component Screening• Contamination• Chromosome Assignment• Suitability (e.g. cancer)

DR

AFT

FIN

Contig-Level Quality

? ?





Changing Technologies, New Resources

Declining clone usage

New technologiesNew public WGS genomes

CostTimeQuality?

Clone-based assembly: 300 Kb interval, 1 contig

WGS-based assembly: 670 Kb interval, 6 contigs

?


GCA_001297185.1



Lander and Waterman

(1988) Genomics

SequencedNot sequenced

1X Coverage

5X Coverage

10X Coverage

37% 63%

0.6% 99.4%

0.005% 99.995%

The likelihood a base is seq’d.Coverage

N50

HuRef

SOAPdenovoNA12878

ALLPATHSNA12878

MHAPCHM1

Chaisson and Eichler (2015)

AK1HX1


Measure of contiguity. Half of the assembly is in contigs this length or greater.

Variant analysis

Annotation

Clinical Diagnostics

Comparative genomics

Transcriptomics

?

Sample/

Population

Assembly level (contig, scaffold,

chromosome)

Full/partial genome

representation

Sequencing method

Assembly method

Contiguity

Coverage

Segmental duplication/

repeat representation

Gene representation

Diploid/

Haploid






De novo assembly assessment

CHM1 and CHM13 Assemblathon• 46 XX haploid hydatidiform moles (U. Surti)• How good are each of the assemblies?• How suited are these assemblies for use in reference

curation? (GRC)

Accession Short Name Sample Submitter Assembler Data Coverage

GCA_001307025.1 CHM1_CA_P6 CHM1 Phillippy CA 8.3rc2 P6 61

GCA_001297185.1 CHM1_FC_P6 CHM1 Chin Falcon 0.3+ P6 61

GCA_000983465.1 CHM13_CA1 CHM13 Phillippy CA 8.3rc2 P5+P6 70




GCA_000983455.2 CHM13_FC CHM13 Chin Falcon 0.4 P5+P6 70

http://www.biorxiv.org/content/early/2016/08/30/072116

Assembly Assessments• General QA (NCBI)

• Assembly stats (length, contiguity)• Annotation• Assembly-assembly alignment to reference• Comparison to BAC inserts • BAC end placements (CHM1 only)

• BioNano map comparison (MGI)• Illumina alignments (Phillippy, Li)

• Quality/Errors• Coverage• Paired end distribution

Resource Sample

Illumina reads CHM1, CHM13

BioNano map CHM1, CHM13

BAC library CHM1, CHM13

BAC library end seqs CHM1

Fingerprint Map CHM1


CHM1 GRCh38 primary CHM1_FC_P6 CHM1_CA_P6

GCF_000001305.14 GCA_001297185.1 GCA_001307025.1

Total Length 3,099,734,149 2,996,426,293 2,939,630,703

Contig N50 56,413,054 26,899,841 20,609,304

Num Ctgs 1,385 3,641 4,850

QV (Koren) ND 44.64 42.28

CHM13 GRCh38 primary CHM13_FC CHM13_CA1 CHM13_CA2 CHM13_CA3 CHM13_CA4

GCF_000001305.14 GCA_000983455.2 GCA_000983465.1 GCA_001015355.1 GCA_000983475.1 GCA_001015385.1

Total Length 3,099,734,149 2,941,135,618 3,061,240,732 3,028,917,871 2,996,416,935 3,065,003,163

Contig N50 56,413,054 10,549,591 13,331,528 19,357,701 5,550,336 12,252,446

Num Ctgs 1,385 4,961 15,538 11,138 10,430 12,091

QV (Koren) ND 43.00 41.21 39.94 42.89 41.36


CHM1 Assemblies CHM13 Assemblies De novo assembly assessment

FRCbam Error Curves

FRCbam C-E Curves

(Sergey Koren)


Assembly Not Aligned

(%)

Split

Alignment

(%)

Coverage

<95% (%)

Dropped

coding

transcripts

Dropped

non-coding

transcripts

Proteins with

frameshifts*

GRCh38

GCA_000001405.15

22 (0.04%) 10 (0.02%) 17 (0.04%) 2 0 19

CHM1_CA_P6

GCA_001307025.1

117 (0.23%) 291 (0.23%) 426 (1.08%) 226 160 983

CHM1_FC_P6

GCA_001297185.1

65 (0.13%) 171 (0.34%) 234 (0.60%) 214 167 1012

CHM13_CA1

GCA_000983465.1

50 (0.10%) 345 (0.68%) 386 (0.98%) 274 213 503

CHM13_CA2

GCA_001015355.1

49 (0.10%) 320 (0.63%) 335 (0.85%) 272 213 439

CHM13_CA3

GCA_000983475.1

46 (0.09%) 616 (1.22%) 632 (1.61%) 240 187 627

CHM13_CA4

GCA_001015385.3

50 (0.10%) 400 (0.79%) 404 (1.03%) 259 197 450

CHM13_FC

GCA_000983455.2

94 (0.18%) 482 (0.96%) 568 (1.44%) 281 202 346

50867 RefSeq transcripts were aligned to each assembly

*GRCh38 frameshifts exclude alternate loci


RefSeq Transcript Analysis

Assembly Not Aligned

(%)

Split

Alignment

(%)

Coverage

<95% (%)

Dropped

coding

transcripts

Dropped

non-coding

transcripts

Proteins with

frameshifts*

GRCh38

GCA_000001405.15

22 (0.04%) 10 (0.02%) 17 (0.04%) 2 0 19

CHM1_CA_P6

GCA_001307025.1

117 (0.23%) 291 (0.23%) 426 (1.08%) 226 160 983

CHM1_FC_P6

GCA_001297185.1

65 (0.13%) 171 (0.34%) 234 (0.60%) 214 167 1012

CHM13_CA1

GCA_000983465.1

50 (0.10%) 345 (0.68%) 386 (0.98%) 274 213 503

CHM13_CA2

GCA_001015355.1

49 (0.10%) 320 (0.63%) 335 (0.85%) 272 213 439

CHM13_CA3

GCA_000983475.1

46 (0.09%) 616 (1.22%) 632 (1.61%) 240 187 627

CHM13_CA4

GCA_001015385.3

50 (0.10%) 400 (0.79%) 404 (1.03%) 259 197 450

CHM13_FC

GCA_000983455.2

94 (0.18%) 482 (0.96%) 568 (1.44%) 281 202 346

50867 RefSeq transcripts were aligned to each assembly

*GRCh38 frameshifts exclude alternate loci


RefSeq Transcript Analysis





Credits

GRCh38 Collaborators• NCBI RefSeq and gpipe annotation team• Havana annotators• Karen Miga• David Schwartz• Steve Goldstein• Mario Caceres• Giulio Genovese• Jeff Kidd• Peter Lansdorp• Mark Hills• David Page• Jim Knight• Stephan Schuster• 1000 Genomes

GRC SAB• Rick Myers• Granger Sutton• Evan Eichler• Jim Kent• Roderic Guigo• Carol Bult• Derek Stemple• Jan Korbel• Liz Worthey• Matthew Hurles• Richard Gibbs

Assemblathon Collaborators• Jason Chin• Adam Phillippy• Sergey Koren• Heng Li

GRCTina Graves-LindsayKaryn Meltz SteinbergKerstin HoweRichard DurbinPaul FlicekLaura ClarkeDeanna ChurchCurators!Developers!

CHM1 Evaluation CHM13 Evaluation


Health & Medicine

AGBT2017 Reference Workshop: Schneider