Strand-seq Core

 For example, Strand-seq has been used for the phasing of genomes and mapping of structural variants in human genomes in studies published in Nature (2,3), Cell (4) and Science (5). In a local collaboration we recently showed that, using a combination of long-read and Strand-seq data from the same person, it is possible to assign a parent of origin to any allele using only a blood sample from the proband (6). For this Parent-of-Origin-Aware genomic analysis (POAga), Strand-seq data is used to generate chromosome-length haplotype information (7) to complement ONT long read whole genome sequence data as well as data about DNA methylation at imprint locations. By combining data from the two different sequencing techniques, any allele on any chromosome in a person can now be assigned to its parent of origin without analysis of parental DNA (6).

Strand-seq library production is currently performed in nanoliter volumes in open nanoliter arrays (8). This advance has resulted in a major increase in the number and quality of Strand-seq libraries that can be produced.

Parent-of-Origin-Aware Genomic Analysis

1. Falconer, E., Hills, M., Naumann, U., Poon, S.S., Chavez, E.A., Sanders, A.D., Zhao, Y., Hirst, M. and Lansdorp, P.M. (2012) DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution. Nat Methods, 9, 1107-1112.
2. Liao, W.W., Asri, M., Ebler, J., Doerr, D., Haukness, M., Hickey, G., Lu, S., Lucas, J.K., Monlong, J., Abel, H.J. et al. (2023) A draft human pangenome reference. Nature, 617, 312-324.
3. Logsdon, G.A., Rozanski, A.N., Ryabov, F., Potapova, T., Shepelev, V.A., Catacchio, C.R., Porubsky, D., Mao, Y., Yoo, D., Rautiainen, M. et al. (2024) The variation and evolution of complete human centromeres. Nature, 629, 136-145.
4. Porubsky, D., Hops, W., Ashraf, H., Hsieh, P., Rodriguez-Martin, B., Yilmaz, F., Ebler, J., Hallast, P., Maria Maggiolini, F.A., Harvey, W.T. et al. (2022) Recurrent inversion polymorphisms in humans associate with genetic instability and genomic disorders. Cell, 185, 1986-2005 e1926.
5. Ebert, P., Audano, P.A., Zhu, Q., Rodriguez-Martin, B., Porubsky, D., Bonder, M.J., Sulovari, A., Ebler, J., Zhou, W., Serra Mari, R. et al. (2021) Haplotype-resolved diverse human genomes and integrated analysis of structural variation. Science, 372.
6. Akbari, V., Hanlon, V.C.T., O’Neill, K., Lefebvre, L., Schrader, K.A., Lansdorp, P.M. and Jones, S.J.M. (2022) Parent-of-origin detection and chromosome-scale haplotyping using long-read DNA methylation sequencing and Strand-seq. Cell Genomics, 100233.
7. Porubsky, D., Sanders, A.D., van Wietmarschen, N., Falconer, E., Hills, M., Spierings, D.C., Bevova, M.R., Guryev, V. and Lansdorp, P.M. (2016) Direct chromosome-length haplotyping by single-cell sequencing. Genome Res, 26, 1565-1574.
8. Hanlon, V.C.T., Chan, D.D., Hamadeh, Z., Wang, Y., Mattsson, C.A., Spierings, D.C.J., Coope, R.J.N. and Lansdorp, P.M. (2022) Construction of Strand-seq libraries in open nanoliter arrays. Cell Rep Methods, 2, 100150

The above sequencing is sufficient for Strand-seq applications such as chromosome-length phasing of haplotypes for POAga and large structural variation detection, but genome assembly studies may require deeper genome coverage. Sample and sequencing costs may vary depending on the desired number of libraries, their desired sequencing depth and the possibility to make and sequence libraries concurrently with samples from different experiments.

The standard Strand-seq package includes cell culture with BrdU, preparation and fluorescence-activated cell sorting (FACS) of fixed nuclei, library preparation using the cellenONE, size selection and quality control of libraries, AVITI Next-generation DNA sequencing, and Strand-seq data processing. For other single-cell whole genome sequencing (WGS) applications, cell culture, nuclei preparation, and FACS can be omitted to reduce costs. Whole blood (2 x 6 mL sodium heparin) or cultured cell lines (frozen in cryovials or in culture media) are acceptable for submission. Alternatively, Coriell catalogued cell lines can be order by the SCORE. The SCORE will provide FastQ files and basic sequencing metrics upon request. We can offer assistance using Strand-seq bioinformatics tools.

Strand-seq Data Production Package

· Cell Culture, Nuclei Preparation & FACS - $200 / sample

· Nanolitre Library Preparation & QC - $100 / sample (in a batch of ~15-20 samples)

· AVITI Sequencing and Data Processing - $1770 (2 x 75 bp) or $2760 (2 x 150 bp) / run (multiplexed pool of ~15-20 samples)

· Labour - $40 / hour

· A surcharge is applied for external users (25%) and corporate users (40%) of the total cost, and may include an additional $500 administrative fee per invoice billed quarterly.

Typical cost per sample (20+ good cells, > 10 million read pairs): $500-1000 depending on pooling of libraries and sequencing strategy.

 Despite different sequencing chemistries, Cloudbreak Freestyle technology allows direct sequencing compatibility with Illumina-based libraries. AVITI sequencing kits can provide over 1 billion read pairs per run with significantly lower costs per Gigabase of data at this scale than standard benchtop Illumina sequencers. The SCORE provides direct AVITI next-generation short read sequencing services for any compatible user sequencing libraries or as a part of the Strand-seq Data Production Package.

AVITI Sequencing

· AVITI 150 cycle High (2 x 75 bp, >900 million Read Pairs, >135 Gb) - $1770 / run

· AVITI 300 cycle High (2 x 150 bp, >900 million Read Pairs, >270 Gb) - $2760 / run

· A surcharge is applied for external users (25%) and corporate users (40%) of the total cost, and may include an additional $500 administrative fee per invoice billed quarterly.

The cellenONE uses gentle acoustic waves to generate droplets, allowing for more gentle isolation of cells for cloning applications with higher cell viability compared to standard flow cytometry cell sorters. Single cells can be isolated into a wide range of formats, including 72-well Terasaki plates, 96-well plates, 384-well plates, microscope slides, or 5184-nanowell Takara ICELL8 chips. This instrument is used for single-cell isolation and nanolitre reagent dispensing for Strand-seq library preparation. Instrument operation is managed by trained SCORE personnel only.

Custom cellenONE Services

· cellenONE Spotting Session - $60 / session

· Labour - $40 / hour

· A surcharge is applied for external users (25%) and corporate users (40%) of the total cost, and may include an additional $500 administrative fee per invoice billed quarterly.

External Collaboration

For collaborators outside UBC and the BC Cancer Agency, service/material transfer agreements need to be set up before samples can be received and processed. Please contact Dr. Peter Lansdorp, using the contact form below, and provide relevant details for collaboration requests.

Workflow

1. Send a message through the online form on this page for any inquiries for Strand-seq or other AVITI and cellenONE services. The SCORE can help determine the best suited conditions and strategies for the application.

2. Complete any statements of work (SOW) or material transfer agreements (MTA) as applicable.

3. When ready to submit samples, detailed sample shipping instructions will be provided. The following sample formats are acceptable:

     a. Sample information for cell line from Coriell to be ordered and shipped by the SCORE directly

     b. Shipped viable cells in culture flasks at room temperature

     c. Shipped viable cells frozen in cryovials on dry ice

     d. Shipped fresh blood sample in 2 x 6 mL sodium heparin tubes at room temperature

4. Demultiplexed FastQ files will be provided after sequencing is complete.

5. Invoices will be sent for billing via wire transfer.

breakpointR - https://github.com/daewoooo/breakpointR 

ASHLEYS QC - https://github.com/friendsofstrandseq/ashleys-qc 

StrandPhaseR - https://github.com/daewoooo/StrandPhaseR 

InvertypeR - https://github.com/vincent-hanlon/InvertypeR