Church sfaf13

Keep CalmAnd

Carry on SequencingDeanna M. Church Staff Scientist, NCBI

@deannachurch

http://genomereference.org

Valerie Schneider, NCBI

Photograph: Paul Popper/Popperfoto/Getty Images

GRCh38 is coming(September, 2013)

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

http://www.bioplanet.com/gcat

Dennis et al., 2012

1q32 1q21 1p21

1p21 patch alignment to chromosome 1


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

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


NM_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


NM_031192.3: transcript from C57BL/6JNM_031193.2: transcript from FVB/N

An assembly is a MODEL of the genome

Assembly Model

BAC insertBAC vector

Shotgun sequence

Assemble

GAPS

Finishing

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

NCBI36 (hg18)

GRC

h37

(hg1

9)

NCBI35 (hg17)

GRCh37 (hg19)

AL139246.20

AL139246.21

Daly et al., 2013

Fixing Rare/Incorrect Bases

GRCh37B Sites for Update: n=1164

Sites with unique successful ctg 1148 (98.6%)

Avg Length 448 bp

Min/Max Success Length 51/791 bp

Avg Coverage 80x

Read Source (all contigs)

High coverage 32%

Low coverage 57%

Exome 10%

Fixing Rare/Incorrect Bases

Build sequence contigs based on contigs defined in TPF (Tiling Path File).

Check for orientation consistenciesSelect switch pointsInstantiate sequence for further analysis

Switch point

Representative chromosome sequence

RP11-34P13 64E8 RP4-669L17 RP5-857K21 RP11-206L10 RP11-54O7

Gaps

NCBI36

nsv832911 (nstd68) Submitted on NCBI35 (hg17)

NCBI35 (hg17) Tiling Path

GRCh37 (hg19) Tiling Path

Gap Inserted

Moved approximately 2 Mb distal on chr15

NC_0000015.8 (chr15)

NC_0000015.9 (chr15)

Removed from assembly

Added to assembly


Sequences from haplotype 1Sequences from haplotype 2

Old Assembly model: compress into a consensus

New Assembly model: represent both haplotypes

AC074378.4AC079749.5

AC134921.2AC147055.2

AC140484.1AC019173.4

AC093720.2AC021146.7

NCBI36 NC_000004.10 (chr4) Tiling Path

Xue Y et al, 2008

TMPRSS11E TMPRSS11E2

GRCh37 NC_000004.11 (chr4) Tiling Path

AC074378.4AC079749.5

AC134921.1AC147055.2

AC093720.2AC021146.7

TMPRSS11E

GRCh37: NT_167250.1 (UGT2B17 alternate locus)

AC074378.4AC140484.1

AC019173.4AC226496.2

AC021146.7

TMPRSS11E2

nsv532126 (nstd37)

Adding Novel Sequence

1000G ph1 decoy sequence, viewed by:• GenBank alignment• Percent Repeat Masker• Repeat Masker type• Sequence Source (HTG, HuRef, ALLPATHS)

Genovese et al., 2013


Karen Hayden and Jim Kent

Human Resolved for GRCh38


Examples

Preview of GRCh38 (scheduled Fall 2013)

TEX28 TKTL1

LOC101060233(opsin related)

LOC101060234(TEX28 related)

GRCh37 (current reference assembly)chrX

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

FAM23_MRC1 Region, chr10

Segmental Duplications

1KG accessibility Mask

Novel Patch 250 kb of artificial duplication

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

GRCh38 is coming(September, 2013)

Church sfaf13

Technology

Transcript of Church sfaf13