Zebra Finch Seg Dup Analysis

1. Genome

2. Parameters for Pipeline

3. Analysis

Zebra Finch Genome

• The Genome (Jul. 2008 assembly of the zebra finch genome taeGut1, WUSTL v3.2.4) is downloaded from UCSU. This assembly was produced by the Genome Sequencing Center at the Washington University in St. Louis (WUSTL) School of Medicine.

• The zebra finch DNA used for the shotgun sequencing and the BAC and cosmid libraries was derived from a single male domesticated zebra finch. The initial assembly was generated using PCAP with approximately 6X coverage. About 1.0 Gb of the 1.2-Gb genome has been ordered and oriented along 33 chromosomes and one linkage group. The chromosome names are based on their homologous chromosomes in the chicken (Gallus gallus).

• Total genome size (gapped) 1,233,186,341 bp

Seg Dup detection pipelines

• WGAC to detect Seg Dup in genomic assemblies by looking for homologouse pairs ( >1 kb in length >90% identity).

• WSSDto detect Seg Dup in given sequences based on depth coverage of WGS (whole-genome shotgun reads). Depth coverage > Average + 3SD. Done by Ginger Cheng.

Parameters and notes for WGAC pipeline

• Repeats– The sequences download from UCSC has been soft masked.

• UCSC rmsk options: RepeatMasker -align -s -species 'Taeniopygia guttata'

– The repeat coordinates were reverse generated based on the soft-masked sequences.

• Blast parsing seeds in WGAC pipeline:– the seed size is 250 bp.

Result from WGAC Pipeline

• Total pairs of WGAC detected (>1 kb and >90% identity) 198180

• Inter chromosome pairs 81415• Intra chromosome pairs 116742• Chromosome inter and intra

(excluding chr_random and chrUn) 26510• ChrUn inter and intra 172670• Total WGAC NR (bp) 384,501,909• Total genome size (with gap) 1,233,186,341

Notes:• The NR space of WGAC is about 31% zebra finch genome, which is too high. It is

either due to the incomplete repeat masking or redundant sequences in chr_random and chrUn. 87% of the total WGAC pairs (inter and intra) have at least one sequence in each pair is on chrUn. The result indicates a big portal of false positive WGAC is from chrUn.

General analysis of WGAC length and identity distribution

1. Length distribution peaked at 1-2 kb, intra > inter, with 87% of WGAC related to chrUn.2. Identity distribution peaked at 97-98%. Few are higher than 99%.

WGAC identity distribution

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Identity

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WGAC length distribution

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General analysis, NR distribution on chromosome high SD in chrUn

None redundant WGAC length distribution on Chromosome

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20000000

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chr1chr1_randomchr1Achr1A

_randomchr1Bchr1B

_randomchr2chr2_randomchr3chr3_randomchr4chr4Achr4A

_randomchr4_randomchr5chr5_randomchr6chr6_randomchr7chr7_randomchr8chr8_randomchr9chr9_randomchr10chr10_randomchr11chr11_randomchr12chr12_randomchr13chr13_randomchr14chr14_randomchr15chr15_randomchr16_randomchr17chr17_randomchr18chr18_randomchr19chr19_randomchr20chr20_randomchr21chr21_randomchr22chr22_randomchr23chr23_randomchr24chr24_randomchr25chr25_randomchr26chr26_randomchr27chr27_randomchr28chr28_randomchrLG

2chrLG

5chrLG

E22

chrLGE

22_randomchrZchrZ

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n

Total (bp)

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Percentage of none redundant WGAC on chromosome

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chr1chr1_randomchr1Achr1A

_randomchr1Bchr1B

_randomchr2chr2_randomchr3chr3_randomchr4chr4Achr4A

_randomchr4_randomchr5chr5_randomchr6chr6_randomchr7chr7_randomchr8chr8_randomchr9chr9_randomchr10chr10_randomchr11chr11_randomchr12chr12_randomchr13chr13_randomchr14chr14_randomchr15chr15_randomchr16_randomchr17chr17_randomchr18chr18_randomchr19chr19_randomchr20chr20_randomchr21chr21_randomchr22chr22_randomchr23chr23_randomchr24chr24_randomchr25chr25_randomchr26chr26_randomchr27chr27_randomchr28chr28_randomchrLG

2chrLG

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22_randomchrZchrZ

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Chromosome

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Global image shows the inter and intra pairs of 10 kb and above 90% in identity without or with chrUn. The red indicates the inter chromosomal pairs and blue indicates intra chromosomal pairs.

Without chrUnWith chrUn

WGAC page

• http://eichlerlab.gs.washington.edu/help/linchen/zfinch/zfinch_wgac.html

WSSD analysis done by Ginger

http://eichlerlab.gs.washington.edu/help/ginger/zebrafinch/ • Downloaded the WGS reads; about 11,683,735 reads from trace archive at NCBI.

• Downloaded zfinch-finished BACs. These BACs are used to determine the threshold for WGS depth coverage. For 5-kb window, the average number of reads is 59. The threshold for 5-kb window is 110, for 1-kb it’s 22.

• Used UCSC taeGut1 database rmsk tables as input to mask the genome for repeats with divergence <=10%.

(UCSC rmsk options: RepeatMasker -align -s -species 'Taeniopygia guttata')

WSSD results

• A total of 16,076 regions with 44,218,871 bp were found in wssdGE10K_nogap.tab (which has a 10-k cut-off). 13,782 of them are on chrUn.

• A summary table of WGAC intersect with WSSD is at http://eichlerlab.gs.washington.edu/help/linchen/zfinch/data/wgacCMPwssd.out.xls

General view showing WGAC (>5kb) and WSSD on all chromosomes

Grey above lines are WSSDBrow below lines are WGAC

Union of WSSD and WGAC gene intersect with Seg Dups

• A nonredundant union of WGAC and WSSD is generated with cut-off size at 10 kb (AllDup10kb.tab). There are 3,839 NR regions with 50,902,487 bp, which is about 10 mb more than WSSD alone.

• However, be aware there may be false positive sites, especially on chrUn, since we know there are high false positive WGACs on chromosomes and chrUn.

Summary table 1

  total chrN chrUnNo. nr

interval file

wssd (bp) 44,218,871 11,237,985 35,080,886 729 wssdGE10K_nogap.tab

wgac (bp) 384,501,909 232,493,308 152,008,601 7387 oo.weild10kb.join.all.cull

AllDup (bp) 394,988,746 235,022,961 159,965,785 5934 allDUP

Wssd and Wgac shared 8,195,577 3,182,128 5,013,449    

Genome (bp) 1,233,186,341 1,057,961,026 175,225,315

Large SDs >=10 kb

• SD >=10 kb in size were pulled out. There are a total of

3,839 intervals with length 50,902,487 bp in the allDup.tab.

The study of the chromosome only WGAC

• The Segment duplications on sequences assigned to chromosome should be more reliable sequences with less artifact.

• It should contains sequences reflecting best of the

assembly.

• Total Dup length 105,145,288 bp• Intra Dup length 100,234,309 bp• Inter Dup length 8,499,428 bp

• More Dup is intra chromosome dup >90%• These intra chromosome dup are predominantly short

range intra dup, see the global view on next slide

Global view of 90%-5k and 94%-5k respectively, showing significant amount of WGAC pairs are intra chromosome short range duplications.

The blowup view showing WGAC on chromosome 1 at 5k and 94%. This is WGAC detected on sequences assigned

to chromosome only

Detail of a sample region on chr1

WSSD

GreyDepth of coverage by reads

Assembly Gaps

Intra chromosome Homology pairs

The average identity for the for the reads mapped to the region.

Red >99%

Orange >98%

Yellow > 97%

Green > 96%

Text description for slide 20

• Each black line represent the chromosome regions as indicated by ticks.• Blue bars and pairs are the intra chromosome homologous pairs (segment

duplications) found.• Red bar and pair on chromosome line represent the inter chromosome

homologous pairs (inter chromosome Segment Duplications).• The grey bars under the chromosome line represent the depth of coverage

at the regions by WGS reads in 1kb window. The longer the bar is , the higher the depth of coverage by sequence reads.

• The color bar under the chromosome line represent the average identity for all the reads mapped to the region. Red(>99%), Orange(>98%), yellow(>97%), green (>96%).

• The black bar above the chromosome line represent WSSD detected.• The purple vertical line on chromosome line represent the assembly gaps.• Each tick represent the 10000bp; each line is 100kb.

result

• Most of the intra chromosomal pairs are very close to each other. In most cases, one sequence within the pair has gaps on both ends, which suggest the contig is not physically connected to its adjacent sequences. It was placed at current position by the mate pairs.

• Some of them are also next to each other, separated by a gap.

• We have not see in sampled region that a single contig contains both sequences within the pairs of intra chromosome segment duplications.

• Consider observation mentioned above, we think there is a high possibility that they could be assembly artifacts introduced by assembler.