Finished sequence and assembly of the DUF1220-rich 1q21 region using a haploid human genome
- Majesta O’Bleness†1,
- Veronica B Searles†1,
- C Michael Dickens1,
- David Astling1,
- Derek Albracht2,
- Angel C Y Mak3,
- Yvonne Y Y Lai3,
- Chin Lin3,
- Catherine Chu3,
- Tina Graves2,
- Pui-Yan Kwok3,
- Richard K Wilson2 and
- James M Sikela1Email author
© O’Bleness et al.; licensee BioMed Central Ltd. 2014
Received: 18 December 2013
Accepted: 6 May 2014
Published: 20 May 2014
Although the reference human genome sequence was declared finished in 2003, some regions of the genome remain incomplete due to their complex architecture. One such region, 1q21.1-q21.2, is of increasing interest due to its relevance to human disease and evolution. Elucidation of the exact variants behind these associations has been hampered by the repetitive nature of the region and its incomplete assembly. This region also contains 238 of the 270 human DUF1220 protein domains, which are implicated in human brain evolution and neurodevelopment. Additionally, examinations of this protein domain have been challenging due to the incomplete 1q21 build. To address these problems, a single-haplotype hydatidiform mole BAC library (CHORI-17) was used to produce the first complete sequence of the 1q21.1-q21.2 region.
We found and addressed several inaccuracies in the GRCh37sequence of the 1q21 region on large and small scales, including genomic rearrangements and inversions, and incorrect gene copy number estimates and assemblies. The DUF1220-encoding NBPF genes required the most corrections, with 3 genes removed, 2 genes reassigned to the 1p11.2 region, 8 genes requiring assembly corrections for DUF1220 domains (~91 DUF1220 domains were misassigned), and multiple instances of nucleotide changes that reassigned the domain to a different DUF1220 subtype. These corrections resulted in an overall increase in DUF1220 copy number, yielding a haploid total of 289 copies. Approximately 20 of these new DUF1220 copies were the result of a segmental duplication from 1q21.2 to 1p11.2 that included two NBPF genes. Interestingly, this duplication may have been the catalyst for the evolutionarily important human lineage-specific chromosome 1 pericentric inversion.
Through the hydatidiform mole genome sequencing effort, the 1q21.1-q21.2 region is complete and misassemblies involving inter- and intra-region duplications have been resolved. The availability of this single haploid sequence path will aid in the investigation of many genetic diseases linked to 1q21, including several associated with DUF1220 copy number variations. Finally, the corrected sequence identified a recent segmental duplication that added 20 additional DUF1220 copies to the human genome, and may have facilitated the chromosome 1 pericentric inversion that is among the most notable human-specific genomic landmarks.
Keywords1q21 DUF1220 domain Hydatidiform mole
A major landmark in the modern era of medical genomics research is the sequence and assembly of the human genome. The current genome build, however, contains numerous gaps and areas of potential misassembly. Completion of an accurate assembly is a continuing challenge given the presence of multiple highly duplicated and complex regions that remain largely intractable to analysis with commonly used assembly techniques . Nonetheless, finishing these regions has significant implications for identifying causative disease loci and in turn efficacious treatments for patients with genetic and genomic disorders . The 1q21 region of chromosome 1 is a classic example, given its association with multiple clinical disorders and its complex architecture, with multiple regions of duplication that make complete assembly extremely difficult. The 2009 human genome assembly reflects this challenge, containing 14 gaps in the 7.7 Mb 1q21.1-2 region.
Closing these gaps in the current 1q21 build is a particularly pressing problem given that recurrent genetic and genomic variations in this region have been implicated in a multitude of disease phenotypes: neuropsychiatric diseases such as autism [3, 4] and schizophrenia [5, 6], microcephaly and macrocephaly [7, 8], cardiac conduction and structural defects [9, 10], multiple congenital anomalies [11–13], and ocular deficits . Additionally, this region contains multiple Neuroblastoma Breakpoint Family (NBPF) genes encoding 238 of the known 270 copies of DUF1220, a protein domain that has undergone a striking copy number increase specifically in the human lineage [14, 15]. While this extreme DUF1220 copy number increase has been linked to the evolutionary expansion of the human brain [16, 17], the many interspersed and tandem DUF1220 paralogs in the 1q21 region are thought to be major contributors to 1q21 genomic instability leading to numerous disorders . Ascertaining the exact involvement of DUF1220 and other 1q21 sequences in these diseases has been hindered by the incomplete nature of the 1q21 assembly and of the DUF1220-encoding gene family (NBPF) in particular. Without a complete, accurate assembly, genotype-phenotype associations are difficult to identify, and those that are found may not provide a complete picture of disease etiology and in some cases may even be incorrect and misleading.
To pursue the completion of the 1q21 genomic region, a haploid hydatidiform mole (CHM1) genome was utilized which reduces the challenges introduced by using a diploid, polymorphic genome . Using bacterial artificial chromosomes (BACs) produced from the CHM1 genome the 14 gaps that remained in this region were closed and a single haploid genomic path generated that spans the 1q21.1-2 region. This new, completed assembly was used to more precisely analyze genomic structural variation in individuals with 1q21 CNVs and microcephaly or macrocephaly.
Results and discussion
Sequence finishing and assembly
Copy number differences in 1q21 between the GRCh37 build and the CHM1 assembly
Total 1q21 DUF1220
Description of NBPF genes in GRCh38 assembly
No. of DUF1220
No. of DUF1220 triplets
Confirmation of DUF1220 Copy Number Estimates
Digital droplet PCR
DUF1220 clade (subtype) copy number estimates were compared within each NBPF gene between the new and old assemblies (Figure 2). DUF1220 domains are subdivided into six clades based on sequence similarity, referred to as conserved (CON) clades 1 through 3 and human lineage specific (HLS) clades 1 through 3. CON1 and HLS1 were analyzed for DUF1220 copy number validation. The CON1 copy number determined by ddPCR of CHM1 DNA was comparable to that seen across multiple control samples, suggesting that the assembly of this clade within NBPF genes accurately reflects the general population. The HLS1 copy number, meanwhile, was slightly lower than that seen in the majority of control samples, suggesting that the CHM1 genome may have fewer HLS copies than would be found in healthy individuals. It should be noted that HLS1 DUF1220 domains are highly polymorphic within the population and as such these results do not necessarily suggest that either version of the 1q21 assembly will be more useful for future analysis of copy number-phenotype correlations for this locus. Copy number measurements of PDE4DIP as measured by ddPCR reflected those predicted by the molar assembly and mirrored those seen in healthy controls. In addition, a primer set mapping uniquely to NBPF11 and NBPF24 (NBPF genes differing by only 3 nucleotides) from the GRCh37 assembly was used to determine if the loss of one of these regions was unique to the CHM1 cell line or representative of healthy individuals as well. ddPCR results demonstrate a single copy, confirming loss of NBPF24 and retention of the one copy of NBPF11. Overall, results indicate that the CHM1 assembly generally is representative of healthy individuals and can be used in place of the current human genome build.
Mapping disease samples
By remapping array results for 40 patients with 1q21 CNVs to the new assembly, regions affected by deletions and duplications were identified with better certainty and precision. In the future, the more accurate and complete 1q21 assembly will also allow for better mapping of breakpoints of these copy number changes, which will in turn aid in localizing disease-causing genes and regulatory loci, and in the development of a morbidity map of the region.
This investigation demonstrated that resources developed from a haploid hydatidiform mole genome could effectively be used to complete assembly of chromosome 1q21, one of the most complex and evolutionarily dynamic regions of the human genome. Completing this region also has significant implications for studying human disease given the numerous disorders associated with CNVs, mutations and chromosomal aberrations in the 1q21 region. Additionally, the complete 1q21 assembly will play an integral role in studies of human evolution, as 1q21 contains the majority of the 289 DUF1220 protein domain copies, 160 of which were added specifically to the human genome since the Homo/Pan divergence . The new 1q21 assembly has already led to the discovery of a novel copy of SRGAP2, a gene in the 1q21 region that may be important for the elaboration of neuronal processes in the human brain . Efforts to localize disease-causing genes and regulatory regions that have previously been hindered by the incomplete nature of the 1q21 assembly and inaccuracies in the region may now move forward with a complete and reliable map to identify causative sequence variations.
Hydatidiform moles are human conception abnormalities that most often arise from the fertilization of an anucleate ovum by a single X-bearing sperm. Subsequent diploidization results in a 46 XX karyotype in which all allelic variation has been eliminated allowing the unambiguous delineation of duplicated DNA as well as haplotype characterization. The hydatidiform mole (CHM1) BAC library (CHORI-17) was previously created by the Children’s Hospital Oakland Research Institute BACPAC Resource by Mikhail Nefedov in Pieter de Jong’s laboratory. The library was prepared from a well-characterized haploid cell line (CHM1htert) from Dr. Urvashi Surti, Director of the Pittsburgh Cytogenetics, laboratory, using the cloning approach described in Osoegawa et al. . This library was used for subsequent analysis.
Sequence finishing and assembly
A minimum tiling path of single haplotype clones was selected based off of a fingerprint map and alignment of existing BAC end sequences (BES) to span the 1q21.1-q21.2 region of interest. Sequences were generated to cover each BAC insert as described below. The clones were pooled prior to sequencing in groups of 25, in equal molar ratio, and a single 454 fragment library and a single 3730 plasmid library were produced. This approach leveraged the high throughput, unbiased 454 data with the 3730 data, which provides long-range linkage, long reads for assembly, and template availability. The 454 pools were sequenced to greater than 25× coverage, and 3730 libraries to a coverage of 4×. In addition, BACs difficult to resolve due to multiple paralog content had individual 3730 libraries created and sequenced.
The data was assembled using a de novo assembly approach using both pcap  and newbler (Roche 454 software package) assembly algorithms to assemble the data. These assemblies were then compared to one another as well as to the human reference sequence, to further guide the assembly and resolve any sequencing ambiguities. This approach has been applied extensively to whole genome bacterial projects in the size range of 5 Mb, with great success, as well as clone pools in human structural variation fosmid projects . An automated improvement process called prefinishing was performed to choose directed work for low quality regions and gaps, and then a manual process of finishing the regions to a level of less than 1 error per 10,000 bp was employed. At the end of this process, a high quality product suitable for identification of sequence differences between the reference sequence and the single haplotype was achieved. The new assembly can be found at accession number JH636052.4.
All DUF1220/NBPF homologous regions were evaluated using the criteria published in O’Bleness et al. . Through a collaboration between the Sikela laboratory, RefSeq at NCBI, and the HUGO Gene Nomenclature Committee, a consensus gene nomenclature was decided upon. This nomenclature is used in the GRCh38 release of the human genome.
Analyses of segmental duplications and DUF1220 HLS triplet expansion events
Evaluation of the 1q21 to 1p11 duplication events was generated by aligning the CHM1 1q21 region to the CHM1 1p11 region using the Exonerate alignment tool with the genome to genome option  and visualized in GBrowse for manual annotation and confirmation. The largest and highest scoring alignments were plotted using the Circos visualization tool (Figure 3) . Evaluation of the relationship between the DUF1220 HLS triplet (hls1-hls2-hls3) sequences in each NBPF gene was performed by aligning each DUF1220 HLS triplet using the PRANK multiple sequence aligner  and generating a phylogenetic tree using the APE package in R (Figure 5) .
Mapping of disease samples to the new 1q21 assembly
40 patient samples from the Baylor College of Medicine with 1q21 microduplications or microdeletions were identified by array comparative genomic hybridization (arrayCGH) using probes specific to the 2009 1q21 assembly. Arrays were previously constructed using Agilent custom array capabilities, designed and processed as described in Dumas et al. . Probes from the initial run based on the old assembly were re-mapped to the new assembly in order to identify loci affected by deletion/duplication status that vary by assembly map.
Droplet digital PCR confirmation of copy number estimates in the new 1q21 assembly
Droplet digital PCR (ddPCR), a third-generation PCR technique , was used to check the copy number estimates of multiple loci in the new assembly. DNA was extracted from CHM1htert cell pellets, provided by Dr. Urvashi Surti, using a Qiagen DNA extraction kit following manufacturer’s protocols. Extracted DNA was digested with the restriction enzyme DDE1. Digested DNA was then added to a PCR mix according to the manufacturer’s protocol including fluorescently tagged probes specific to the region of interest (separate reactions for conserved clade 1 (CON1) (Left ‘AATGTGCCATCACTTGTTCAAATAG’ , Right – ‘GACTTTGTCTTCCTCAAATGTGATTTT’ , Hyb – ‘CATGGCCCTTATGACTCCAACCAGCC’), human lineage specific clade 1 (HLS1) (Left – ‘GCTGTTCAAGACAACTGGAAGGA’, Right - ‘GGGAGCTGCTGGAGGTAGT’ , Hyb – ‘AGAGCCTGAAGTCTTGCAGGACTCAC’), PDE4DIP (Left – ‘GCCTTATTAGCATCCCAAGACAA’ , Right – ‘CCCTGAACAGCCTTTCCTTCT’ , Hyb – ‘CATGCTGTGAAGAAGTCGGTCTACCCCAC’), and a unique region mapping to NBPF11 (Left - ‘GGAAAGTCGGGTTTGTGAGA’, Right – ‘TGGCACAACATCCTGGAATA’ , Hyb – ‘ACAACAGAGGAGAGCGGAGA’) and to a reference sequence of known copy number, RPP30 (Left – ‘GATTTGGACCTGCGAGCG’, Right – ‘GCGGCTGTCTCCACAAGT’ , Hyb – ‘TTCTGACCTGAAGGCTCTGCGC’). Oil droplets containing this mixture were produced using a BioRad droplet generator, resulting in over 14,000 droplets per well. Droplets were then subject to a thermocycling protocol with an annealing temperature of 56°C and read single-file on a droplet reader to compare fluorescence of the target and reference in each droplet. Results were merged to produce a final copy number estimate and this estimate was compared to that provided by the new assembly and to ddPCR results examining the same loci in healthy controls from the Coriell dataset.
Confirmation of NBPFdata using Irys technology
The Irys platform automates high-resolution genome mapping by imaging labeled single DNA molecules in nanochannels. Three hydatidiform mole BACs (CH17-112A12, CH17-353B19 and CH17-382H24) containing NBPF12, NBPF10 and NBPF15, respectively, were selected for genome mapping to validate the sequence assembly of the chr1q21 region. BAC DNA was extracted with Large Construct kit (Qiagen, Valencia, CA) and 300 ng of purified BAC DNA were used for nicking and labeling according to the irysPrep protocol (BioNano Genomics, San Diego, CA). In brief, 300 ng of purified BAC DNA were incubated in a 10 μL nicking reaction at 37°C with 7 U Nt.BspQI (NEB, Ipswich, MA) for two hours followed by heat inactivation of the nicking enzyme at 80°C for 20 minutes. Five microliters of labeling master mix, consisting of 1.5× labeling buffer, 1.5× labeling mix (BioNano Genomics) and 1 U Taq polymerase (NEB), was added to the heat-inactivated nicking reaction mixture and this labeling reaction mixture was incubated at 72°C for an hour. The nicked and labeled DNA was repaired by PreCR® repair mix that contained 10 mM dNTP mix (NEB). One microliter of stop solution (BioNano Genomics) was added to stop the repair reaction. Lastly, the nicked, labeled and repaired DNA was stained overnight with DNA stain (BioNano Genomics). The nicked, labeled, repaired and stained BAC DNA samples (20 μL each) were combined before they were loaded on the IrysChip for genome mapping on the Irys system (BioNano Genomics).
Image detection, genome map alignment, and assembly were performed using software tools developed in-house and packaged into IrysView at BioNano Genomics. Briefly, the DNA backbone and fluorescent labels were detected, integrated, and converted into single-molecule genome maps. De novo assembly of genome maps was performed using a graph-based assembler. Consensus genome maps were then aligned to an in silico map based on the 1q21 sequence assembly.
Array comparative genomic hybridization
Domain of unknown function 1220
Neuroblastoma breakpoint family
Human genome version 19
Genome Reference Consortium Human Build 37
Bacterial artificial chromosome
Droplet digital PCR
Conserved clade 1
Phosphodiesterase 4D-interacting protein
Human lineage specific clades 1/2/3
Children’s Hospital Oakland Research Institute-17 (name of Hydatidiform Mole BAC Library)
BAC end sequences
Washington University St. Louis.
This work was supported by grants 1R01 MH081203-01A2 (JMS), 3R01 MH081203-02S1 (JMS) and R01 HG005946 (PYK).
The authors would like to acknowledge Drs. S.W. Cheung, S.S.C. Nagamani and A. Erez from the Baylor College of Medicine for providing 1q21 deletion and duplication samples, and Dr. Ernest T. Lam at BioNano Genomics for help with data analysis and editing.
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