Abstract
The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.
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Gene Expression Omnibus
Sequence Read Archive
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Acknowledgements
We wish to recognize BGI-Shenzhen's sequencing platform for generating the data in this study. We thank the faculty and staff at BGI-Shenzhen who contributed to this project especially to H. Jiang and B. Wang, who helped on the experimental part, D. Cao, L. Lin and M. Li, who helped on the validation part, and L. Goodman, W. Chen and H. Jia, who provided critical comments on the manuscript. This work was supported by the State Key Development Program for Basic Research of China-973 Program (no. 2011CB809202); the Shenzhen Municipal Government of China (no. JC201005260191A); International Science and Technology Cooperation Program of Shenzhen (GJHZ20130417140835564); Tianjin Municipal Science and Technology special funds for Enterprise Development (no. 14ZXLJSY00320). G.W. was also supported by Alberta Innovates Technology Futures.
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Ju.W., G.W., Yi.L. and H.C. managed the project. Ju.W., G.K.-S.W., Yi.L., H.C., H.W., R.L. and S.H. designed the analyses. Ha.Y., construct the fosmid library. R.L., Y.S., Y.X., Bi.L., G.H., W.H., Bo.L., Z.H. and Yu.L. conducted assembly and evaluation; H.C., H.W., S.H., Y.S., X.T., H.Z., J.L., Y.W., F.Y., P.S., Si.L., P.G., H.H., J.S., D.C., L.C.A.M.T., X.L., Q.F., X.X., X.Z., L.B., K.K., R.D., S.D., R.N., So.L., Ji.W. and Hu.Y. performed the data analyses. H.C., H.W. and S.H. did most of the writing with contributions from all authors.
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Integrated supplementary information
Supplementary Figure 1 Number and length distributions of the inferred fosmids.
Fosmid were inferred by mapping each pool’s sequence reads to hg19. We retained only those reads that mapped to within a 3~50 kb region. (a) Fosmid number in each pool. On average, there were ~32 fosmids per pool. (b) Fosmid size. The average length was 36.8 kb.
Supplementary Figure 2 Fosmids physical coverage distribution.
Blue curve denotes the theoretical coverage distribution, at an average coverage of 8x, and red curve denote the actual coverage. The average fosmid coverage was 8x, with a median of 7x. About 7% of YHref was not covered by fosmids, which may be due to a bias in the fosmid library construction and/or sequencing.
Supplementary Figure 3 Completeness of assembled sequence in each fosmid pool.
The horizontal axis represents the percentage of the fosmid sequence that was assembled in each pool. The vertical axis represents the proportion of fosmid pools at that given percentage. In total, 88.5% of the assembled pools contained at least 80% of the fosmid sequence, and 53.2% of the assembled pools contained at least 95% of the fosmid sequence.
Supplementary Figure 4 Contiguity of assembled sequence for individual fosmids.
The horizontal axis represents the ratio of the longest assembled sequence vs the inferred length of each defined fosmid. The vertical axis represents the proportion of fosmids at the given ratio. 54.7% of fosmids had a longest assembled sequence equal to, or longer than, half of the fosmid length. About 18% of the fosmids were completely assembled.
Supplementary Figure 5 Construction of the haplotype-resolved sequence.
The top (orange) bar represents the non-phased YHref sequence and the bottom (multi-color) bar represents the haplotype-resolved output. The middle (blue) bars represent the fosmid assembled haploid (FAH) sequences belonging to the same haplotype.
Supplementary Figure 6 Theoretical N50 length of haplotype phasing and long homozygous region.
a. Long homozygous regions (>=20 kb) for different populations in 1000 genomes project. Asians have more long homozygous region than other populations. This might be why YH had a shorter haplotype N50 than other individuals sequenced at a comparable fosmid depth. b. The theoretical N50 length distribution of haplotype phasing using the method of the current study, in 4 different individuals. Heterozygous marker numbers are shown at the top-left. The haplotype N50 of YH is expected to be 510 kb with a fosmid coverage of 4x per haplotype (or 8x for a 3 Gb genome).
Supplementary Figure 7 HDG coverage on hg19 and RefSeq genes.
Our HDG sequence was aligned to the hg19 genome using Lastz. Coverage of the chromosomes and gene regions was calculated. “Both” means covered by the two assembled haplotypes (blue), “Single” means covered by just one assembled haplotype (red). a. Coverage information for each chromosome. b. Proportion of RefSeq gene at given coverage.
Supplementary Figure 8 Length distributions of insertions and deletions.
a. Length distribution of short indels (<10 bp). Peaks at multiples of 3 bp in the exon distribution are expected because they do not disturb the reading frames. b. Length distribution of long indels (100 bp~1 kb). The peak at ~300 bp is due an enrichment for Alu element insertions and deletions. Note that there is no bias between insertion and deletion, which is progress compared to previous studies. c. Distribution of long indels (100 bp~1 kb) in unique versus repeat regions. As expected, there are more indels in the repeat regions and the peak at ~300 bp is more pronounced. d. Length distribution of homozygous and heterozygous long indels (100 bp~1 kb).
Supplementary Figure 9 SNP detection and intersection from different methods/platforms.
A total of ~4.0 M SNPs were detected by three different methods/platforms. The majority (68.2%) of these was consistent between all three datasets. However, there were still tens of thousands of methods/platforms specific calls.
Supplementary Figure 10 Indel detection and intersection from different platforms.
We show the number of small indels detected by each method/platform and their intersection, at a flank size of 50 bp. For the ~1 M indels detected, there was only 27.6% concordance.
Supplementary Figure 11 Example of a heterozygous deletion located inside a gene.
This heterozygous deletion was detected by the ASV method but difficult to find by either WGS resequencing method. The yellow block in the reference is the region that was missing from hap2. Below are the WGS reads aligned to this region. This 151 bp deletion covered the 5-UTR and a part of exon1 for the gene PSMD1.
Supplementary Figure 12 Example of a heterozygous insertion located inside a gene.
This heterozygous insertion was detected by the ASV method but difficult to find by either WGS resequencing method. The yellow block in hap1 is the region that was missing from the reference. Below are the WGS reads aligned to this region. Near the breakpoint there were very few reads, perhaps because the insert sequence influenced the alignment. This 54 bp insertion covered exon3 of the gene LATS2.
Supplementary Figure 13 Variation rate for YH vs hg19 and heterozygosity between the two haplotypes of YH.
The curves at the top and the right summarize the distribution of heterozygosity rates for the two haplotypes of YH and the variation between YH and hg19, respectively. The black line indicates the 99% cutoff for each distribution.
Supplementary Figure 14 The classification of novel gene sequences.
a. Classification of different types of novel and gap covered sequences. i) novel insertion; ii-iv), novel haplotypes; v-vii), gap covered sequences; viii), orphan scaffolds. b. Distribution of novel sequences based on their length and number, in 100 bp bins. Novel sequences of length >1000 bp accounted for 93% of the total length. The longest was 123 kb. c. Distribution of breakpoints for novel sequences. Most of the novel sequences were in non-coding (intron, repeat and intergenic) regions. Only 0.8% were in CDS regions. These distributions are subdivided by the length of the sequence, represented by the color bars. d. Repeat content based on RepeatMasker.
Supplementary Figure 15 Examples of cis- and trans-acting genes.
a. Cis-acting gene DSPP on 4q22.1 encoding dentin sialophosphoprotein. Mutations in DSPP are associated with Dentinogenesis imperfecta, Shields type II, and deafness. b. Trans-acting gene CA9 on 9p13.3. Diseases associated with mutations in CA9 include horseshoe kidney and renal cell carcinoma. GO annotations include carbonate dehydratase activity.
Supplementary Figure 16 Allele specific methylation and expression.
Venn diagram showing the relationship between allele specific methylation (ASM) and allele specific expression (ASE). The numbers refer to the gene count. The red/brown circle inside the larger ASM circle represents genes where ASM was detecting in the promoter region.
Supplementary Figure 17 Construction of the fosmid libraries.
Approximately 30 fosmid clones were cultured together to form a single fosmid pool. Then, 3 μg of DNA from each pool was digested, and fragments with insert size ranging from 180 to 800 bp were selected. Adapters containing the 11 bp barcode were ligated to these selected fragments to form a single pooled-fosmid library. Barcoded fragments from 60~320 single pooled-fosmid libraries were pooled again (evenly) to create a Stage I barcode library. DNA fragments of sizes between 180 bp to 650 bp (lengths exclude barcode) from each Stage I barcode library were used to construct two independent libraries (one with small insert sizes and one with intermediate insert sizes). Each library was then PCR amplified with index primers, each of which contained an 8 bp barcode, to form a Stage II barcode library.
Supplementary Figure 18 Indel positional concordance as a function of flank size for the different methods of detection.
To determine the best flank size for use in indel detection, we plotted the concordance between the ASV and resequencing based analyses. The results stabilize at above 50 bp.
Supplementary Figure 19 Length distributions for method-specific short indels.
Short indels (1-50 bp) detected only by one method/platform, were selected out and plotted according to the length. Top-right figure provided information for indels with length between 10 and 50.
Supplementary Figure 20 Example of ASV-specific indel supported by fosmid aligned reads.
This was a 3 bp heterozygous deletion in a region covered by fosmids from eight independent pools, two of which supported the deletion.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–20 and Supplementary Tables 1–3 and 5 (PDF 1737 kb)
Supplementary Table 2
Genes in hypervariable regions (XLSX 49 kb)
Supplementary Table 3
Annotation of predicted novel genes (XLSX 12 kb)
Supplementary Table 4
Cis and Trans genes annotation (XLSX 21 kb)
Supplementary Table 5
ASE and ASM gene analysis (XLSX 247 kb)
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Cao, H., Wu, H., Luo, R. et al. De novo assembly of a haplotype-resolved human genome. Nat Biotechnol 33, 617–622 (2015). https://doi.org/10.1038/nbt.3200
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DOI: https://doi.org/10.1038/nbt.3200
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