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Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube

Abstract

Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here we demonstrate barcode partitioning of long DNA molecules in a single compartment using “on-bead” barcoded tagmentation. The key to the method that we call “contiguity preserving transposition” sequencing on beads (CPTv2-seq) is transposon-mediated transfer of homogenous populations of barcodes from beads to individual long DNA molecules that get fragmented at the same time (tagmentation). These are then processed to sequencing libraries wherein all sequencing reads originating from each long DNA molecule share a common barcode. Single-tube, bulk processing of long DNA molecules with 150,000 different barcoded bead types provides a barcode-linked read structure that reveals long-range molecular contiguity. This technology provides a simple, rapid, plate-scalable and automatable route to accurate, haplotype-resolved sequencing, and phasing of structural variants of the genome.

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Figure 1: Summary of the bead-based indexing workflow and the intra- vs.
Figure 2: Detection of deletion and interchromosomal translocation using linked read information.

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Acknowledgements

We would like to thank the research, development, and software engineering departments at Illumina for sequencing technology development. Specifically, we would like to thank C.L. Pan at Illumina for the sequencing technology development and G. Bean, J. Leng, and S. Swamy at Illumina for data analysis software development. We would like to thank R. Daza for providing HeLa and NA12878 Gentra DNA preparations. The genome sequence described in this paper was derived from a HeLa cell line. Henrietta Lacks, and the HeLa cell line that was established from her tumor cells in 1951, have made significant contributions to scientific progress and advances in human health. We are grateful to Henrietta Lacks, now deceased, and to her surviving family members for their contributions to biomedical research.

Author information

Authors and Affiliations

Authors

Contributions

F.J.S., K.L.G., and N.G. conceived the study; F.J.S. and M.C.R. oversaw the technology development. F.Z. and D.P. led the assay development, L.C., J.T., S.J.N., and D.P. performed the experiments, and analyzed the data. F.Z. performed the phasing analysis and wrote custom analysis software. A.G., E.J., R.J., and N.M. helped with the assay development. Y.Z., M.W., and E.W. prepared the bead pool. A.H. developed the data analysis pipeline. M.R. led the project coordination. F.J.S., F.Z., L.C., K.L.G., D.P., and J.S. co-wrote the paper. All authors contributed to the revision and review of the manuscript.

Corresponding author

Correspondence to Frank J Steemers.

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Competing interests

The authors declare competing financial interests in the form of stock ownership, patents, or employment through Illumina, Inc.

Integrated supplementary information

Supplementary Figure 1 Sequencing overview.

(a) Example sequencing plot for the intensity versus cycle (IVC) of the library from the hybrid method. (b) Example plot for the IVC plot with the single-tube method. The first 15 cycles of read 1 and read 2 show the typical Tn5 insertion bias42.

Supplementary Figure 2 Typical example of a BioanalyzerTM library size distribution.

Supplementary Figure 3 Island structure visualization.

An 868 kb genomic region is shown in the top panel for the reads sharing the same barcode. The reads are grouped into two clusters called island, which originate from two long DNA molecules interacting with beads having the barcode 1. The zoomed in snapshot shows the reads distribution within island2 with a total genomic coverage of around 25%.

Supplementary Figure 4 Proximal versus distal read distribution from the hybrid method using GentraTM DNA.

Reads with the same barcode are aligned to the genome. The genomic distance between adjacent reads sharing the same barcode shows a bimodal distribution. The short distances between the adjacent reads within the island structure contribute to the proximal peak centered around 2 kb. On average, there are about 500-1000 long DNA molecules transposed by multiple beads with the same barcode. These long DNA molecules are randomly distributed across the genome, generating a distal peak for the distance between adjacent islands around 3Gbp/(500 or 1000) ~3-6Mbp.

Supplementary Figure 5 The workflow for phasing and structural variant analysis.

The Input unphased VCF files of the genomes used in this study were obtained from a previous publication. Raw sequencing reads were demultiplexed by their unique barcode and partitioned into individual fastq files. Reads for each partition were then aligned to the reference genome and mapped to discrete islands. The islands that covered at least two heterozygous variants were retained for phasing. Islands from all partitions were subsequently combined. The genome then was split into partitions such that no informative islands (islands covering more than one heterozygous SNP) overlapped with the partition boundaries. For each partition, we used H-BOP to generate the phasing blocks. The phased SNPs, combined with island structure generated before, were used as input for the structural variant analysis.

Supplementary Figure 6 Phasing yield and accuracy.

Phasing yield (a) and accuracy (b) for GentraTM prep NA12878 using the hybrid method. Phasing yield (c) and accuracy (d) for freshly prepared NA12878 using the single-tube method. The inset in the panel is a zoomed in representation of the phasing yield/accuracy from 0-50 kb.

Supplementary Figure 7 Comparison of island lengths between GentraTM prep and freshly prepared DNA.

Same number of reads are randomly subsampled from the original data to generate the histograms.

Supplementary Figure 8 The histogram of the number of reads per barcode for the 150,000 combinatorial bead pool using the single-tube method.

Supplementary Figure 9 Detection of heterozygous deletion using linked reads. Each color represents a distinct barcode and the short vertical bar represents a mapped read.

Supplementary Figure 10 Confirmation of heterozygous deletion detection using trio analysis.

The sequencing depth for NA12878, NA12891, and NA12892 were normalized around the deletion locations. One of the parents showed lower depth of sequencing in the same region indicative of a heterozygous deletion.

Supplementary Figure 11 Interchromosomal translocation detection for the Hela genome. 17 out of total 20 interchromosomal translocations previously reported37 show strong signal in the barcode sharing heat map.

Supplementary Figure 12 Haplotyping accuracy at the island level.

The analysis was applied to 5000 randomly selected barcodes. The number of islands versus the hetSNPs per islands follows the expected exponential decay as larger islands are underrepresented (blue bars). Edit errors per number of hetSNPs in an island are depicted as red diamonds. Although not applied in this paper, conflicting islands can be filtered out before input into the H-BOP.

Supplementary Figure 13 Comparison of genomic coverage uniformity for Zheng et al8, single-tube method, hybrid method, and Truseq PCR-free.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 (PDF 1672 kb)

Supplementary Table 1

The number of reads with different barcodes in intra- vs inter- beads transposition experiment (XLSX 7 kb)

Supplementary Table 2

The DNA sequences of the indexed transposons, the oligos on beads, the sequencing primers and the primers for deletion detection. (XLSX 41 kb)

Supplementary Table 3

Comparison of coverage and SNP/INDEL recall/precision among different approaches. (XLSX 10 kb)

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Zhang, F., Christiansen, L., Thomas, J. et al. Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube. Nat Biotechnol 35, 852–857 (2017). https://doi.org/10.1038/nbt.3897

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