BRIEF RESEARCH REPORT published: 09 January 2020 doi: 10.3389/fgene.2019.01315 Flow Sorting Enrichment and Nanopore Sequencing of Chromosome 1 From a Chinese Individual Lukas F. K. Kuderna 1†*, Manuel Solís-Moruno 1,2†, Laura Batlle-Masó 1,2†, Eva Julià 3,4†, Esther Lizano 1†, Roger Anglada 2, Erika Ramírez 4, Alex Bote 4, Marc Tormo 2,5, Tomàs Marquès-Bonet 1,6,7,8,9, Òscar Fornas 4,10†* and Ferran Casals 2†* Edited by: 1 Institut de Biologia Evolutiva, CSIC-Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona (PRBB)", Youri I. Pavlov, Barcelona, Spain, 2 Genomics Core Facility, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, University of Nebraska Medical Center, Parc de Recerca Biomèdica de Barcelona (PRBB), Barcelona, Spain, 3 Serveis Científico-Tècnics, Institut Hospital del Mar United States d'Investigacions Mè diques (IMIM), Barcelona, Spain, 4 Flow Cytometry Unit, Centre for Genomic Regulation (CRG), The Reviewed by: Barcelona Institute for Science and Technology (BIST), Barcelona, Spain, 5 Scientific IT Core Facility, Departament de Satomi Mitsuhashi, Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona (PRBB), Yokohama City University, Barcelona, Spain, 6 CNAG-CRG, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Japan Barcelona, Spain, 7 Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), Barcelona, Spain, Yael Michaeli, ́ ̀ 8 Institucio Catalana de Recerca i Estudis Avanç ats (ICREA), Barcelona, Spain, 9 Institut Catala de Paleontologia Miquel Crusafont, Tel Aviv University, Universitat Autò noma de Barcelona, Barcelona, Spain, 10 Flow Cytometry Unit, Departament de Ciències Experimentals i de la Salut, Israel Universitat Pompeu Fabra (UPF), Parc de Recerca Biomèdica de Barcelona (PRBB), Barcelona, Spain *Correspondence: Lukas F. K. Kuderna Sorting of individual chromosomes by Flow Cytometry (flow-sorting) is an enrichment lukas.kuderna@upf.edu Òscar Fornas method to potentially simplify genome assembly by isolating chromosomes from the oscar.fornas@upf.edu context of the genome. We have recently developed a workflow to sequence native, Ferran Casals unamplified DNA and applied it to the smallest human chromosome, the Y chromosome. ferran.casals@upf.edu † Here, we modify improve upon that workflow to increase DNA recovery from These authors have contributed equally to this work chromosome sorting as well as sequencing yield. We apply it to sequence and assemble the largest human chromosome - chromosome 1 - of a Chinese individual Specialty section: using a single Oxford Nanopore MinION flow cell. We generate a selective and highly This article was submitted to Genomic Assay Technology, continuous assembly whose continuity reaches into the order of magnitude of the human a section of the journal reference GRCh38. We then use this assembly to call candidate structural variants against Frontiers in Genetics the reference and find 685 putative novel SV candidates. We propose this workflow as a Received: 18 September 2019 Accepted: 02 December 2019 potential solution to assemble structurally complex chromosomes, or the study of very Published: 09 January 2020 large plant or animal genomes that might challenge traditional assembly strategies. Citation: Keywords: chromosome enrichment, nanopore sequencing, chromosome sequencing, chromosome sorting, flow Kuderna LFK, Solís-Moruno M, karyotyping, structural variation, genome assembly Batlle-Masó L, Julià E, Lizano E, Anglada R, Ramírez E, Bote A, Tormo M, Marquès-Bonet T, Fornas O and Casals F (2020) Flow Sorting INTRODUCTION Enrichment and Nanopore Structural genetic variation is abundant and has important functional impact (Conrad et al., 2010). Sequencing of Chromosome 1 From a Chinese Individual. A human genome has been estimated to harbor more than 2,000 structural variants (SV), which are Front. Genet. 10:1315. typically defined as variants that affect at least 50 bp (Mills et al., 2011). They include balanced doi: 10.3389/fgene.2019.01315 (inversions, translocations) and unbalanced forms (insertions, deletions, duplications) (Mills et al., Frontiers in Genetics | www.frontiersin.org 1 January 2020 | Volume 10 | Article 1315 Kuderna et al. Chromosome Sorting for Nanopore Sequencing 2011). The functional impacts are mainly produced by altering was replaced by sodium citrate and sodium sulfite to a final the number of copies of coding sequences, and thus the gene concentration of 10 and 25 mM respectively and incubated at expression levels, or disrupting coding or regulatory regions with least 2 hours to enhance peak resolution in the flow karyotype. a potential effect not only in the closer genes but also extending Purification and concentration were carried out as previously to hundreds of kilobases away (Weischenfeldt et al., 2013). SVs described with the exception that after SPRI bead purification have been associated both to Mendelian and common disorders, DNA was eluted in 20 µl of Low TE buffer. Libraries for although it is difficult to exactly define the phenotypic impact due sequencing were prepared from the purified DNA following to the presence of many functional regions in the same variant, as the protocol of the Ligation Sequencing Kit SQK-LSK 109 well as variable expressivity and penetrance even across family (Oxford Nanopore Technologies). A 48 hours MinION run (Weischenfeldt et al., 2013). Nevertheless, and despite of its high was performed in a FLO-MIN106 flow cell. abundance and potential impact, the study of SVs has been less accelerated in comparison to single nucleotide variants and short Assembly, Error Correction, and SV Calling insertions and deletions (indels). It has been mainly limited by We called bases from the raw fast5 signal data using Guppy (v. the short reads generated by massive parallel sequencing 2.2.2) with the following command line: technologies and the relatively low coverage in large guppy_basecaller -i $input -s $output – sequencing efforts (e.g., 1,000 Genomes Project) (Huddleston flowcell FLO-MIN106 –kit SQK-LSK109 -t 4 and Eichler, 2016). Also, determining the exact position and –disable_pings mechanism of origin of SVs is not straightforward often due to the presence of terminal repetitive sequences and recurrence, and We mapped the base called reads onto GRCh38 using Minimap2 can be especially challenging in complex structural variants with (Li, 2018) with the ont preset. We sorted the mappings and more than two breakpoints and overlapping or nested converted them to bam using samtools (Li et al., 2009): rearrangements (Collins et al., 2017; Stephens et al., 2018). All this makes difficult the generation of systematic catalogues of SVs minimap2 -x map-ont -t8 -a hg38.fa and the estimation of allelic population frequencies. basecalled_reads.fq| samtools sort -@8 -O BAM The recently emerging possibility to obtain reads of up to of - -o mapped_reads.bam several Megabases in length on the Oxford Nanopore platform We unsuccessfully tried to assemble the raw reads into contigs represents an important advance for the study of structural using canu (v. 1.8) (Koren et al., 2017) with default parameters variants and genome assembly, as it greatly simplifies them assuming a “genome” size of 250 Mb. This command used more (Giordano et al., 2017; Payne et al., 2019). These sequencing than 15 Tb of disk space and did not finish to yield a successful technologies can be combined with enrichment strategies, from assembly on our systems. To overcome the issue, we extracted capture by hybridization to Cas9 based methodologies, to restrict mappings on chromosome 1 and assembled only those: the analysis to specific regions also increasing the sequencing yield. Chromosome isolation is an alternative enrichment canu -p HG00542-chr1 -d HG00542-chr1 strategy which will better maintain molecular integrity with the genomeSize = 250m -nanopore-raw potential of generating longer sequence reads (Jiang et al., 2015; basecalled_reads.chr1_mappings.fq Kozarewa et al., 2015; Gabrieli et al., 2018). We corrected errors in the resulting contigs with Nanopolish We recently developed a workflow to isolate and sequence native (v. 0.11.0) (Simpson et al., 2017). To this end, we remapped the flow-sorted human Y chromosomes on an Oxford Nanopore raw reads to the assembly as shown above. We then went on to MinION device (Kuderna et al., 2019). We sought to apply this create a read db with nanopolish, and split the assembly into method to other chromosomes to generate a population specific chunks of 500 Kb with nanopolish_makerange.py and called the long read assembly, namely for a chromosome 1 of a Chinese variants of each chunk with nanopolish variants individual. We show the generalizability and improve the protocol in terms of DNA recovery and sequencing yield. nanopolish_makerange.py –segment-length 500000 –overlap-length 1000 HG00542- HG00542- chr1.contigs.fasta | xargs -i echo nanopolish METHOD variants –ploidy 2 –consensus -o {}.consensus.round1.vcf -w {} -r Chromosome Isolation and Sequencing basecalled_reads.fq -b HG00542- HG00542- Chromosome preparation, staining, sorting, DNA purification, chr1.contigs.self-mappings.bam -g HG00542- concentration and sequencing were performed as previously chr1.contigs.fasta | sh described in (Kuderna et al., 2019) with some modifications (see supplementary methods). Briefly, chromosomes were We then incorporated the corrections into the assembly: prepared from a lymphoblastoid cell line derived from a nanopolish vcf2fasta -g HG00542- Chinese individual (Coriell, cat. no. HG00542) by using a chr1.contigs.fasta *vcf. polyamine isolation method. Modifications: hypotonic solution was incubated at 37°C for 20 min and polyamine isolation buffer We aligned the resulted polished assembly to GRCh38 chr1 was incubated on ice for 30 min. Additionally, potassium citrate with MUMmer (Kurtz et al., 2004) Frontiers in Genetics | www.frontiersin.org 2 January 2020 | Volume 10 | Article 1315 Kuderna et al. Chromosome Sorting for Nanopore Sequencing nucmer -maxmatch -l 100 -c 500 hg38. Figure 2). The average coverage on chromosome 1 was 28.4 fold, chr1.toplevel.fa. HG00542-chr1.contigs. the coverage on the remaining chromosomes ranged from 0.7 fold polished.fasta -prefix HG00542. on chrX to 2.1 fold on chr19 (Figure 1). This amounts to an 8-fold polished.r1.vs.hg38_chr1 enrichment over a random sampling from bases along a diploid male genome (see Supplementary Table 5). All other We fed the resulting alignments to Assemblytics to obtain chromosomes are depleted from the data, with depletion ranging candidates for SV from 0.27 fold on chr4 to 0.61 fold on chrY. We find the average Assemblytics HG00542.polished.r1.vs.hg38_ depletion on non-target chromosomes to be more efficient in this chr1.delta HG00542.polished.r1.vs. dataset compared to our previous effort on the Y chromosome (0.42 hg38_chr1.10kanchor.50kmax 10000 fold versus 0.61 fold). Nevertheless, we observe the enrichment on bin/Assemblytics/ the target chromosome to be less efficient compared to the Y chromosome. This fact is likely attributable to the more We generated an additional callset with Sniffles (v. 1.0.8) challenging physical separation of chromosome 1 in a human (default parameters), using the previously mapped reads from flow karyogram, as the chromosome clusters are not as well minimap2. For downstream analysis we only retained calls defined as e.g. the one of chrY (Supplementary Figure 1). annotated as “precise” by Sniffles: We assembled the raw data using Canu (Koren et al., 2017). To this end, we removed reads that do not map to GRCh38 chr1 to ease s n i ffl e s - m m a p p e d _ r e a d s . b a m - v the computational load of the assembly (see Method). While this sniffles_callset.vcf. might confound the assembly in regions of large insertions or We filtered all calls that fall within 2 Mb of distance to the translocations, it significantly eases computational burden. We centromere or telomeres. polished remaining single base substitution and indel errors in the resulting assembly with Nanopolish (Simpson et al., 2017). The Comparative Repeat Annotation final assembly has a length of 227.8 Mb and consists of 154 contigs We ran repeatmasker (v. 4.0.7) with the same parameters on both with an N50 of 10.5 Mb. We aligned our assembly to GRCh38 our assembly and GRCh38 to create comparable annotations: chromosome 1, whose total resolved sequence length (i.e. excluding “N” from the assembly) is 230.5 Mb. We find 98.8% of our assembly RepeatMasker -e ncbi -pa 8 -s -species human to cover 97.6% of the reference (Figure 2, Supplementary -no_is -noisy -dir. -a -gff -u assembly.fa Table 4). The boundaries of our contigs are enriched in We calculated the divergence of each repeat to its consensus segmental duplications and satellite repeats in the reference. We using the “calcDivergenceFromAlign.pl” utility included in the observe the highest degree of fragmentation around the RepeatMasker package. centromeric region, which is littered with satellite repeats and segmental duplications, and therefore particularly challenging to assemble. Contigs mapping to these regions also show a drop off in identity to the reference. The centromere on chromosome 1 of RESULTS AND DISCUSSION GRCh38 is an 18 Mb long heterochromatic expansion flanked by segmental duplications that is still unresolved, as in most other We sorted 10 million individual chromosomes 1 from a human chromosomes (Jain et al., 2018). lymphoblastoid cell line derived from a Chinese individual (HG00542) to obtain 5 mg of DNA from a total of 205x106 cultured cells from six independent experiments (see Supplementary Figure 1). Of that, we used 2 million sorted chromosomes theoretically corresponding to 1mg of DNA (Gribble et al., 2009). From this material, we constructed a library using an Oxford Nanopore ligation kit and ran a single MinION flow cell on it. Given limitations in DNA recovery from flow-sorted material we have previously encountered, we have made adjustments to the sorting and purification protocol (see methods and supplementary methods). The higher DNA recovery and higher loading amount on the flow cell yielded between 20 and 131 times more data from a single run than our previous efforts, meaning that a single flow cell is now sufficient to assemble the largest human chromosomes after flow-sorting it. These differences are likely also attributable to improvements in the pore chemistry and base-calling algorithms. After base calling with Guppy, we were left with 2.5 million reads summing to 14.3 Gb of data with a read length N50 of 15.4 Kb. Of them, 10.6 Gb mapped readily to FIGURE 1 | Fold coverage per chromosome. The sequencing is selective for GRCh38, and 5.6 Gb to chromosome 1 (see Supplementary chromosome 1, with all other chromosomes being depleted from the data. Frontiers in Genetics | www.frontiersin.org 3 January 2020 | Volume 10 | Article 1315 Kuderna et al. Chromosome Sorting for Nanopore Sequencing FIGURE 2 | Dot-plot of HG00542 assembly versus GRCh38 chromosome 1. FIGURE 3 | Comparative repeat landscapes of GRCh38 chromosome 1 and The chromosomes are laid out on the respective axis and a dot denotes HG00542 chromosome 1. We find equal resolution across most repeat classes. aligned sequence between the two assemblies. Bars at the height of 250 Mb on the Y axis show the positions of segmental duplications in GRCh38. The assembly is largely colinear to the reference. The large black block in the which used short-read data to detect SVs with several different center of the dot-plot delimits the 18 Mb centromere of chromosome 1. algorithms. This study detected 4,653 SVs on chr1 among 2,504 individuals. Unsurprising, given the technological differences To assess repeat resolution, we produced a comparative repeat between the two datasets, we find comparatively little overlap annotation between our assembly and GRCh38 using repeatmasker between the two call sets with 466 SVs that overlap over 40% in (Smit et al.). We find both assemblies to have very similar either of them. We calculated the frequencies of overlapping SVs in proportions annotated as repetitive overall and for all given each of the superpopulations of the 1,000 genomes data. After repeat families. We then calculated the divergence of all removing variants with an allele count of 2 or less, and multiallelic annotated repetitive elements to their consensus sequence to positions we find these SVs to reach the highest frequencies in east create “repeat landscapes”. We find these landscapes to be highly Asian populations (20.5%); South Asian and American populations similar between the two assemblies. We measured repeat resolution exhibit similar frequencies (18.8% and 18.4%) followed by in our assembly as the proportion of bases annotated as a given European (14.7%) and lastly African (9.8%) populations (see repeat type. We find them to be of comparable quality across all Figure 5 and Supplementary Figures 9–12). We additionally major repeat types, with centromeric & telomeric satellite sequences contrasted our calls with more recently generated ones that also constituting an exception (Figure 3, Supplementary Figure 3, used long-read assemblies for detection (Audano et al., 2019). Supplementary Table 1). Among 15 individuals included in that study there are 6,646 SVs We then used the assembly to generate a call set of candidate structural variants (SVs) against GRCh38. To this end, we aligned our assembly to the reference using MUMmer (Kurtz et al., 2004) and looked for patterns of SVs using Assemblytics (Nattestad and Schatz, 2016). We additionally ran an orthogonal detection approach by mapping the raw reads to GRCh38 and running Sniffles (Sedlazeck et al., 2018). To minimize erroneous calls, we excluded putative SVs within 2 Mb of the centromeric and telomeric regions, as the higher degree of segmental duplications and assembly fragmentation is more likely to yield false positive calls (Audano et al., 2019). By this means, we identified 1,325 SVs with Assemblytics and 940 with Sniffles along chromosome 1. We find 405 of the calls to intersect between the two sets, with 61.4% and 56.9% to be unique to Assemblytics and Sniffles, respectively (see Supplementary Figures 4–8). Of the intersecting calls, we find 230 to lie within genic regions, and 8 to affect the coding portions of the gene (Figure 4 and Supplementary Tables 2–3, 6–7). We sought to assess novel SVs on the one hand, and population frequencies of SVs that might have previously been described in FIGURE 4 | Size distribution of SV calls from both Assemblytics and Sniffles other datasets. To this end, we contrasted our calls against those at different resolutions. Both call sets have clear peaks around 300 bp corresponding to Alu-elements. generated by the 1,000 genomes consortium (Sudmant et al., 2015), Frontiers in Genetics | www.frontiersin.org 4 January 2020 | Volume 10 | Article 1315 Kuderna et al. Chromosome Sorting for Nanopore Sequencing along chr1. We find 291 SVs from our call set to overlap those. The DATA AVAILABILITY STATEMENT calls by Audano et al. include a Chinese individual (HX1) and one from Korea (AK1) (Seo et al., 2016; Shi et al., 2016). Among the The sequencing data has been deposited at the European overlapping SVs, we find 108 deletions (or 44%) and 32 insertions Nucleotide Archive (ENA) under the accession PRJEB34445. (64%) to also be present in both these individuals. Lastly, we identify The assembly can be accessed under GCA_902652775.1. 685 novel SV candidate loci that have not previously been described in neither of the above datasets. In summary, we generated a highly continuous and selective ETHICS STATEMENT assembly of the largest human chromosome from a Chinese Ethical review and approval was not required for the study on individual from flow-sorted native DNA. We show that human participants in accordance with the local legislation and increased efficiency in DNA recovery from flow-sorted institutional requirements. Written informed consent for chromosomes, as well as improvements in nanopore technology, participation was not required for this study in accordance allow for single chromosome assemblies from a single MinION with the national legislation and the institutional requirements. flow cell and that as little as 28-fold coverage is sufficient to yield an assembly with a contig N50 over 10 Mb. As with previous reports, we still find room for improvement in terms of base AUTHOR CONTRIBUTIONS accuracy. We observe a deletion bias in our data, which we find to be twice as frequent as insertions. However, given the constant LK, MS-M, LB-M, TM-B, OF, and FC conceived the project. LK, development in both pore design and base calling algorithms, MS-M, LB-M, EJ, EL, OF, and FC designed the study. LK, MS-M, these issues are likely to improve in the near future. LB-M, and MT performed the bioinformatic analysis. EJ, EL, RA, It is worth noting that flow-sorting chromosomes only ER, and AB performed the experimental analysis. All the authors constitutes a viable approach if the species' chromosomes are participated in the analysis of the data. LK, MS-M, LB-M, EJ, OF, sufficiently distinct in terms of size and GC content. As an and FC wrote the manuscript. example, human chromosomes 9–12 have size differences of up to 6%. However, with our approach, they are hardly distinguishable by flow karyotyping because of similar GC-content across them. FUNDING Conversely, human chromosomes 1–2 have a size difference of only This study was funded by grants RTI2018-096824-B-C22 from the 1.6%. Nevertheless, they differ more strongly in GC content, making Agencia Estatal de Investigación-Ministerio de Ciencia, Innovación them clearly distinguishable by flow karyotyping. Addressing this y Universidades (Spain) and FEDER (EU) to OF and FC, SAF2015- “sortability” of a species' genome is achieved empirically. While 68472-C2-2-R from the Ministerio de Economía y Competitividad assembling mammalian genomes has become routine, there is still a (Spain) and FEDER (EU) to FC, the Centro de Excelencia Severo large amount of plants and animals for which traditional whole- Ochoa, and by Direcció General de Recerca, Generalitat de genome shotgun assembly methods might be computationally Catalunya (2017SGR-702). TM-B is supported by BFU2017- prohibitive given their massive genome sizes. We expect 86471-P (MINECO/FEDER, UE), U01 MH106874 grant, assembling flow-sorted chromosomes to be a viable alternative in Howard Hughes International Early Career, Obra Social “La these cases. Caixa” and Secretaria d'Universitats i Recerca and CERCA Programme del Departament d'Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). LK is supported by an FPI fellowship associated with BFU2014-55090-P (MINECO/ FEDER, UE) and by an EMBO Short-Term Fellowship STF-8286. LB-M is supported by a Formació de personal Investigador fellowship from Generalitat de Catalunya (2018_FI_B00072). MS-M is supported by the María de Maetzu Programme (MDM- 2014-0370-16-3). ACKNOWLEDGMENTS We thank Núria Bonet and Raquel Rasal (Servei de Genòmica UPF) for their help during the library preparation and sequencing. SUPPLEMENTARY MATERIAL FIGURE 5 | Population frequencies of SV discovered in our assembly that overlap calls by the 1,000 genomes project. We find these SVs to be most The Supplementary Material for this article can be found online frequent in East Asian populations, followed South Asian and American, at: https://www.frontiersin.org/articles/10.3389/fgene.2019. European, and lastly African populations. 01315/full#supplementary-material Frontiers in Genetics | www.frontiersin.org 5 January 2020 | Volume 10 | Article 1315 Kuderna et al. Chromosome Sorting for Nanopore Sequencing REFERENCES Li, H. (2018). Minimap2: pairwise alignment for nucleotide sequences. 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(2009). original author(s) and the copyright owner(s) are credited and that the original The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078– publication in this journal is cited, in accordance with accepted academic practice. No 2079. doi: 10.1093/bioinformatics/btp352 use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Genetics | www.frontiersin.org 6 January 2020 | Volume 10 | Article 1315