The Human Reference AssemblyDeanna M. Church Staff Scientist, NCBI

@deannachurch Short Course in Medical Genetics 2013

Updating the assembly

Oh No! Not a new version of the human genome!

Updating the assembly

Updating the assembly

120 Fix PATCHES: Chromosome update in GRCh38

71 Novel PATCHES: Additional sequence added

(adds >5 Mb of novel sequence to the assembly)

(adds >800K of novel sequence to the assembly)

Releasing patches quarterly

GRCh37.p13(160 regions: >3% of chromosomes)

Summer of 2013

Assembly (e.g. GRCh37.p5)

Primary Assembly

Non-nuclear assembly unit

(e.g. MT)

ALT 1

ALT 2

ALT 3

ALT 4

ALT 5

ALT 9

ALT 6

ALT 7ALT

8

PAR

…

Genomic Region(MHC)

Genomic Region

(UGT2B17)Genomic

Region(MAPT)

Patches

Genomic Region(ABO)

Genomic Region(SMA)

Genomic Region

(PECAM1)

Data Model

GCA_000001405.6 /GCF_000001405.17

Primary Assembly

GCA_000001305.1/GCF_000001305.13

ALT 1

GCA_000001315.1/GCF_000001315.1

ALT 2

GCA_000001325.1/GCF_000001325.2

ALT 3

GCA_000001335.1/GCF_000001335.1

ALT 4

GCA_000001345.1/GCF_000001345.1

ALT 5

GCA_000001355.1/GCF_000001355.1

ALT 6

GCA_000001365.1/GCF_000001365.2

ALT 7

GCA_000001375.1/GCF_000001375.1

ALT 8

GCA_000001385.1/GCF_000001385.1

ALT 9

GCA_000001395.1/GCF_000001395.1

Patches GCA_000005045.5GCF_000005045.4

Non-nuclear assembly unit

(e.g. MT)

GCA_000006015.1/GCF_000006015.1

Data Model

GRCh38 is coming(September, 2013)

http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes

http://genomereference.org

Why does missing sequence matter?

GRCh37

Sample genomeDuplicon A Duplicon B

Duplicon A

May or may not detect increased coverage depending on sequencing depthand library quality (easier to find with new technologies than with old, low through technologies)

x x

G>A (allelic difference – true variant)

G>C (paralogous sequence variant- false positive)

http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes

CDC27

1KG Phase 1 Strict accessibility mask

SNP (all)

SNP (not 1KG)

Sudmant et al., 2010

Kidd et al, 2007 APOBEC cluster

Part of chr22 assembly

Alternate locus for chr22

White: InsertionBlack: Deletion

http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes

Mouse Ren1 chr1 (CM000994.2/NC_000067.6): 133350674-133360320

129S6/SVEvTac tiling path

Alignment to C57BL/6J chr1

B6 Genes

129S6/SvEvTac Genes

+ 32Kb in 129S6/SvEvTac

Mouse Ren1 chr1 (CM000994.2/NC_000067.6): 133350674-133360320NM_031192.3: transcript from C57BL/6JNM_031193.2: transcript from FVB/N

129S6/SvEvTac Alt Locus Alignment (allelic)

FVB/N Transcript Alignment (paralog)

129S6/SvEvTac Ren1

FVB Ren2 Tx

Paralogousdiff

SNP +Paralogous

diff

Mouse Ren1 chr1 (CM000994.2/NC_000067.6): 133350674-133360320NM_031192.3: transcript from C57BL/6JNM_031193.2: transcript from FVB/N

Hydin: chr16 (16q22.2)Hydin2: chr1 (1q21.1)Missing in NCBI35/NCBI36 Unlocalized in GRCh37 Finished in GRCh38

Alignment to Hydin2 Genomic, 300 Kb, 99.4% ID

Alignment to Hydin1 CHM1_1.0, >99.9% ID

Alignment to Hydin2 Genomic, 300 Kb, 99.4% ID

Alignment to Hydin1 CHM1_1.0, >99.9% ID

Doggett et al., 2006

Dennis et al., 2012

1q32 1q21 1p21

1p21 patch alignment to chromosome 1

Preview of GRCh38 (scheduled Fall 2013)

TEX28 TKTL1

LOC101060233(opsin related)

LOC101060234(TEX28 related)

GRCh37 (current reference assembly)chrX

http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/issue_detail.cgi?id=HG-21

NCBI36 (hg18)

GRCh

37 (h

g19)

NCBI35 (hg17)

GRCh37 (hg19)

AL139246.20

AL139246.21

Fixing Rare/Incorrect Bases

Fixing Rare/Incorrect Bases

GRCh37B Sites for Update: n=1164Sites with unique successful ctg 1148 (98.6%)Avg Length 448 bpMin/Max Success Length 51/791 bpAvg Coverage 80x

Read Source (all contigs)High coverage 32%Low coverage 57%Exome 10%

Fixing Rare/Incorrect Bases

A = 0.000G=1.000

rs4732519

rs4732519

RP11 WGS reads

Private RP11 variant?Missing in 1000G?

FAM23_MRC1 Region, chr10

Segmental Duplications

1KG accessibility Mask

Novel Patch 250 kb of artificial duplication

Genovese et al., 2013

Adding Novel Sequence

Adding Novel Sequence

Karen Hayden and Jim Kent

Human Resolved for GRCh38

http://genomereference.org

Richa Agarwala

MHC Alternate locus

Alignment to chr6

Making the assembly accessible to existing tools: masking

Query set: 439,109,084 NA12878 HiSeq reads

Masking effectively blocks alignments in regions with high identity

Simulated reads from GRCh37.p9• Unpaired reads• 101 bp• 1x coverage• Default wgsim parameters

Masking parameters• Percent Id: 100%• Step size: 5 bp• Minimum length: 101 bp• Center SNPs in unmasked regions

Masking improves alignments in regions with alternate loci or patches

NA12878 reads whose best alignment was on an alt/patch in the masked assembly were evaluated for their alignment location when aligned to the primary assembly alone

Masking effectively reduces the increase in NA12878 reads that have alignments with MAPQ=0 that occurs when the full assembly is used as an alignment substrate

Take home messages

The assembly you use for analysis is an important part ofyour analysis package. The reference assembly is not a set of linear sequences butcan now represent allelic diversity

Tools still need to catch up. The human reference assembly is updating soon!(Remember: assemblies are not static if you are lucky!)