DOI: 10.5061/dryad.mcvdnck6h
title: Dataset: T-DNA characterization of genetically modified 3-R-gene late blight resistant potato events with a novel procedure utilizing the Samplix Xdrop® Enrichment Technology

description: Before commercialization of genetically modified crops, the events
 carrying the novel DNA must be thoroughly evaluated for agronomic,
 nutritional, and molecular characteristics. Over the years, Polymerase
 Chain Reaction-based methods, Southern blot, and short-read sequencing
 techniques have been utilized for collecting molecular characterization
 data. Multiple genomic applications are necessary to determine the insert
 location, flanking sequence analysis, characterization of the inserted
 DNA, and determination of any interruption of native genes. These
 techniques are time-consuming and labor-intensive, making it difficult to
 characterize multiple events. Current advances in sequencing technologies
 are enabling whole genomic sequencing of modified crops to obtain full
 molecular characterization. However, in polyploids, such as the tetraploid
 potato, it is a challenge to obtain whole genomic sequencing coverage that
 meets regulatory approval of the genetic modification. Here we describe an
 alternative to labor-intensive applications with a novel procedure using
 Samplix Xdrop® enrichment technology and next-generation Nanopore
 sequencing technology to more efficiently characterize the T-DNA
 insertions of four genetically modified potato events developed by the
 Feed the Future Global Biotech Potato Partnership: DIA_MSU_UB015,
 DIA_MSU_UB255, GRA_MSU_UG234 and GRA_MSU_UG265 (derived from regionally
 important varieties Diamant and Granola). Using the Xdrop® /Nanopore
 technique, we obtained a very high sequence read coverage within the T-DNA
 and junction regions. In three of the four events, we were able to use the
 data to confirm single T-DNA insertions, identify insert locations,
 identify flanking sequences, and characterize the inserted T-DNA. We
 further used the characterization data to identify native gene
 interruption and confirm the stability of the T-DNA across clonal cycles.
 These results demonstrate the functionality of using the Xdrop® /Nanopore
 technique for T-DNA characterization. This research will contribute to
 meeting regulatory safety and regulatory approval requirements for
 commercialization with small shareholder farmers in target countries
 within our partnership.
description: Plasmid and T-DNA Materials The plasmid
 pSIM4392 was developed by Simplot Plant Sciences (Boise, ID). The genetic
 elements within the T-DNA are in the supplementary data (S2. Table 1). To
 summarize, pSIM4392 has a T-DNA that contains four cassettes. The first
 cassette (elements 5 to 11, S2. Table 1) contains the selectable marker
 <em>nptII</em> gene and the expression of the gene confers
 kanamycin resistance used for the selection of plants containing the
 T-DNA. The second cassette (elements 13-15, S2. Table 1) contains
 <em>Rpi-vnt1</em> (<em>vnt1</em>) gene from
 <em>Solanum venturi </em>(Foster, S.J., 2009). The third
 cassette (elements 17-19, S2. Table 1) contains
 <em>Rpi-mcq1</em> (<em>mcq1</em>) gene from
 <em>Solanum mochiquense</em> (Aguilera-Galvez, C., 2020). The
 fourth cassette (elements 21-23, S2. Table 1) contains
 <em>Rpi-blb2</em> (<em>blb2</em>) gene from
 <em>Solanum bulbocastanum</em> (van der Vossen, E.A., 2005).
 The gene products from the last three cassettes, VNT1, MCQ1 and BLB2, are
 R-proteins involved in the plant immune response that protects potato from
 foliar late blight infection caused by <em>P. infestans</em>
 (Jones, J.D. and Dangl, J.L., 2006). These genes are in the CC-NB-LRR
 (coiled-coil, nucleotide-binding, leucine-rich repeat) class of resistance
 (R) genes (Paluchowska, P., et.al (2022). Each cassette is a cisgene
 expressed under its native promoter and terminator, pVnt1 and tVnt1 for
 <em>Rpi-vnt1</em>, pMcq1, and tMcq1 for
 <em>Rpi-mcq1</em>, pBlb2 and tBlb2 for
 <em>Rpi-blb2</em>.  The sequence of pSIM4392 plasmid can be
 found in the Dryad Dataset (Zarka, KA., 2023). A map of the entire
 pSIM4392 plasmid is shown in Figure 1. Plant
 Materials Potato plant events were produced using
 Agrobacterium transformation as part of the Global Biotech Potato
 Partnership and by collaboration with Simplot Plant Sciences (Boise,
 Idaho).  The C58-derived Agrobacterium strain AGL1 (Lazo et al., 1991)
 carrying pSIM4392, was used to transform potato internode explants
 following the method described by Richael et al. (2008). A flowchart
 highlighting the development and selection of lead potato events
 transformed with T-DNA in plasmid pSIM4392 is shown in the supplementary
 material (S1 Figure 2). Transformed internode explants were regenerated on
 medium containing 150 mg/l kanamycin to select for lines containing a
 T-DNA insert. The pSIM4392 backbone contained the <em>isopentenyl
 transferase</em> (<em>ipt</em>) gene. Events expressing
 the <em>ipt</em> gene will have a cytokinin phenotype (stunted
 growth) or have atypical phenotypes such as elongated trichomes or
 chlorotic leaves. (Kunkel et al., 1999). They would have also transferred
 some or all of the plasmid backbone. These events were eliminated from
 further analysis. For both the Diamant and the Granola host varieties
 around 300 events were advanced to analyze T-DNA copy number. The T-DNA
 copy number was determined by digital droplet Polymerase Chain Reaction
 (ddPCR) according to the protocol in Collier et al. (2017). Events with
 more than one copy were eliminated from further analysis.  Internal
 regions of the T-DNA were tested in the events with Polymerase Chain
 Reaction (PCR) analysis and any negative events were eliminated. R-gene
 function was tested in growth chamber plant pathology bioassays and in
 field trials (unpublished, Douches, D., 2023). The plant events, selected
 as the lead events, and used in this study, are DIA_MSU_UB015 and
 DIA_MSU_UB255 from the host variety Diamant, and GRA_MSU_UG234 and
 GRA_MSU_UG265 from the host variety Granola. These events will be referred
 to as UB015, UB255, UG234 and UG265 respectively.    
 Genomic DNA Isolation For PCR analysis and
 ddPCR analysis, genomic DNA was isolated from leaf tissue using the DNeasy
 Plant Mini Kit: CAT#69,104 (Qiagen) according to the manufacturer’s
 instructions. DNA isolation for the Xdrop® enrichment technology method
 required high molecular weight genomic DNA. High molecular weight DNA is
 essential to obtain the long sequencing reads that will span not only the
 flanking region of the insert location but also the T-DNA. DNA isolation
 was done with leaf tissue of greenhouse-grown plants and an isolation
 procedure modified from Saghai-Maroof et al. (1984). Fresh leaf tissue (2
 g) was ground with a mortar and pestle in 7 ml of extraction buffer (0.1 M
 Tris, pH 8.0/1.4 M NaCl/0.02M EDTA/2% hexadecyltrimethylammonium bromide/
 1% 2-mercaptoethanol). Transfer of further DNA-containing solutions was
 only done with universal pipet tips with wide tip openings (USA
 Scientific, Ocala, FL). The ground leaf tissue mixture was filtered
 through 2 layers of cheesecloth and incubated at 65°C for 30 min with
 occasional gentle mixing. An equal volume of chloroform/isoamyl alcohol
 24:1 (vol:vol), was added, and the solution was mixed by inversion to form
 an emulsion that was centrifuged at 3000 rpm for 10 min at room
 temperature. The aqueous phase was removed, and 2/3 vol of isopropanol was
 added and mixed by gentle inversions. The precipitated DNA was washed with
 1ml 70% ethanol and then dissolved in 300 ml of resuspension buffer (10mM
 Tris 1 mM EDTA). The DNA samples were evaluated for DNA size distribution
 by capillary electrophoresis on a Tapestation<sup>TM</sup>
 instrument, using Genomic DNA ScreenTape (Agilent Inc., Santa Clara, CA)
 according to the manufacturer’s instructions. The DNA samples were then
 shipped to Samplix (Denmark). Inserted T-DNA
 analysis In polyploids, such as the tetraploid potato,
 it is difficult to have the coverage in whole genomic sequencing needed to
 meet regulatory review recommendations.  Samplix developed an enrichment
 instrument technology called the Xdrop ®, which enables targeted DNA
 fragments to be encapsulated and enriched so they can be sequenced using
 next-generation sequencing. As mentioned in the introduction, Blondal et
 al. (2021), previously described identifying flanking regions of inserted
 T-DNA. Here we describe utilizing the technology to achieve high sequence
 coverage across the entire T-DNA region of each of the lead 3R-gene late
 blight resistant events as well as the identification of flanking regions
 on either side of the T-DNAs. A.
 Xdrop® enrichment technology
 The Xdrop® enrichment technology uses the Xdrop® instrument,
 cartridges, and reagents along with the DNA samples of interest.  The
 workflow includes two parts: 1. Primer design, enrichment and
 quantification. 2. Digital Polymerase Chain Reaction (dPCR) Generation,
 Sorting of Xdrop® droplets, Droplet Multiple Displacement Amplification
 (dMDA) and Evaluation of Enrichment. A graphic of the workflow was
 previously described in Blondal et al. (2021) and included here in the
 supplementary material (S1 Figure 1).<strong>
 </strong>  1. Primer design, enrichment and
 quantification The DNA samples were evaluated for size
 distribution and quality by Tapestation<sup>TM</sup> System
 (Agilent Technologies Inc.), using Genomic DNA ScreenTape according to the
 manufacturer’s instructions. Primer sets for enrichment and quantification
 were designed specifically for the detection of sequences within the
 insert site.  This was done to achieve coverage across the entire T-DNA
 and obtain genomic flanking sequence data as well.  The primers were
 tested and successfully implemented to enrich two Regions of Interest
 (ROIs) and are listed in the supplementary material (S2. Table 2). Primer
 set, ROI1_8F and ROI1_8R, are located within the 3’ region of the
 <em>Rpi</em>-<em>mcq1</em> gene. The primer set
 ROI2_9F and ROI2_9R set is located within the 3’ region of the
 <em>Rpi-vnt1</em> gene. The highest amount of enrichment will
 occur in the sequence surrounding the ROIs. Therefore, to ensure that
 high-quality sequence data can be achieved after the enrichment and span
 the entire T-DNA the ROIs are located near the center of the T-DNA.  Assay
 evaluation of primers described in the supplementary material (S2. Table
 2). was performed by using quantitative polymerase chain reaction (qPCR)
 as previously described in Blondal et al. (2021). The
 DNA samples were purified using HighPrep™ PCR Clean-up Bead System
 according to the manufacturer’s instructions (MAGBIO Genomics) with the
 following changes. Bead-to-Sample ratios were 1:1 (vol:vol) and elution
 was performed by heating the sample in the elution buffer for 3 minutes at
 55 °C before separation on the magnet. The samples were eluted in 20 μl 10
 mM Tris pH8. Purified DNA samples were quantified by Quantus (Promega
 Inc.) Fluorometer<sup>TM</sup>, according to the
 manufacturer’s instructions. 2. dPCR Generation,
 Sorting of Xdrop ® droplets, Droplet Multiple Displacement Amplification
 (dMDA) and Evaluation of Enrichment The dPCR
 generation, sorting of Xdrop® droplets and droplet Multiple Displacement
 Amplification (dMDA) were performed as previously described in Blondal et
 al. (2021). In short, millions of double emulsion droplets were generated
 by the Xdrop ® instrument, followed by droplet PCR (dPCR), which was
 conducted by taking each DNA sample and compartmentalizing the DNA into
 droplets that included dPCR master mix and ROI primer sets.  After droplet
 production, the DNA within the droplets was subjected to PCR amplification
 using the enrichment primers, described above, to generate droplets
 carrying the ROI. The resulting enrichment of the ROI targets was
 evaluated by quantitative Polymerase Chain Reaction (qPCR) according to
 the Xdrop ® manufacturer instructions. After the dPCR protocol, the
 droplets were collected and dyed to generate a fluorescent signal in
 droplets carrying the ROI. The positive droplet populations were sorted
 from the negative droplets using a fluorescence-activated cell sorting
 (FACS) instrument, specifically, a SONY benchtop SH800S cell sorter with a
 100 μm nozzle (Sony Biotechnology). A more detailed description of the
 process is presented in the Xdrop ® manufacturer instructions. DNA from
 the positive droplets was released and re-encapsulated into single
 emulsion droplets by Xdrop ® and subjected to multiple displacement
 amplification (dMDA) according to Xdrop ® manufacturer instructions. The
 amplified DNA was isolated and quantified. The dMDA reactions were then
 diluted in molecular grade H<sub>2</sub>O (1:9 vol/vol) and
 subjected to qPCR reaction using validated qPCR assays. 10 ng of target
 DNA was used as a control as well as the dMDA controls for background and
 contamination evaluation. Enrichment Calculation Tool (https://samplix.com/calculations/per-amountof-genetic-material/actual-enrichment-calculator) was used to calculate the enrichment of the dMDA samples according to the Xdrop® manufacturer instructions. Sample Cycle Threshold (CT) values in Real-time Polymerase Chain Reaction (RT-PCR) and the clusters with the FACS analysis were evaluated according to the Xdrop® manufacturer instructions. B. Nanopore Genomic Sequencing and Bioinformatic Analysis Minion Oxford Nanopore Sequencing platform was used to generate long-read sequencing data from the dMDA samples as described by the manufacturer’s instructions (Premium whole genome amplification protocol (SQK-LSK109) with the Native Barcoding Expansions 1-12 and 13-24 (EXP-NBD104/114)). For each of the events, 1.2 μg amplified of dMDA DNA was treated with T7 Endonuclease I followed by size selection, end-repair, barcoding, and adaptor ligation using the Oxford Nanopore Technology (ONT) Ligation Sequencing Kit. After library generation the sample was loaded onto a GridION flow cell R9.4.1 (20 fmol) and run for 16–24 hours under standard conditions as recommended by the manufacturer (Oxford Nanopore Inc.). To get sequences with high accuracy the generated raw data files (fasta5) were base called using Guppy 5.0.17 with super high accuracy and quality 10 filtering. Using the sequence data obtained and the potato reference genome <em>Solanum tuberosum</em> DM1-3 PGSC v4.04 pseudomolecules downloaded from http://spuddb.uga.edu/index.shtml (SPUD DB Potato Genomics Research, 2023), the T-DNA, was mapped to the genome with Minimap2.  Using the bedtools bamtobed utility, a bed file was created, corresponding to the mapping regions. After masking those regions, using the bedtools maskfasta utility, a new reference genome was created.  The T-DNA only region of the pSIM4392 sequence was then added as an extra chromosome.  All reads were then mapped to the new masked genome, containing the T-DNA, using Minimap2 with default settings for Oxford Nanopore reads (-ax map-ont).  The sequence viewer Integrative Genomics Viewer (IGV) was used to examine the coverage profile for the T-DNA. Both primary and supplementary mapping reads were extracted, that map to the T-DNA by using the utilities samtools view and seqkit grep. These are the reads of interest for finding the location of the T-DNA insertions in the genome. Using the reference genome and the extracted reads from the T-DNA, Minimap2 was used to map the reads from the T-DNA to the masked genome which includes the T-DNA. Using the samtools view utility, a TAG was added containing the read name to the bam file, followed by using the bedtools genomecov utility to generate a list of areas with T-DNA coverage in the bam file. The IGV viewer was then used to identify the insertion borders. All sequences from each event were compared with the plasmid T-DNA to determine the integrity of the T-DNA sequences for each event. The workflow for characterizing the T-DNA in a GM event after Xdrop® enrichment and ONT sequencing is described in Figure 2. A table of the software, tools and resources used is available in the supplementary material (S2. Table 3). Flanking sequence analysis and Sanger Sequencing The reads mapping to the construct were mapped back to the genome to identify the sequence that would span the breakpoint between insert and genome.  These sequences were then used to identify the chromosomal location of the insert at each the left border and right border T-DNA.  The identified insert site was confirmed by PCR across left and right breakpoints between insert and genome followed by Sanger sequencing. The PCR primers and conditions are found in the supplementary material (S2. Table 4)<strong>. </strong>The confirmational PCR analysis and Sanger Sequencing covered the junction as well as 1000bp of the flanking regions. Sanger Sequencing was conducted by submitting amplicon DNA and primers to The Research Technology Support Facility (RTSF) at Michigan State University. Insertion Analysis in non-GM varieties The sequence of the potato reference genome <em>Solanum tuberosum</em> DM1-3 PGSC v4.04 pseudomolecules downloaded from http://spuddb.uga.edu/index.shtml could be different than the genome sequence of the host varieties of our events.  Therefore, to further characterize the insert location, the sequence of the genomic insertion site in the non-GM variety of each event was determined using primers that hybridize to the flanking genomic regions of the insertion sites identified in the flanking sequence analysis.  Primers and PCR conditions for this analysis can be found in the supplementary material (S2. Table 5). PCR amplification resulted in amplicons that covered the insert loci in the non-GM variety of each event. Each of the resulting fragments was ligated into the TOPO-TA plasmid using Invitrogen™ TOPO™ TA Cloning™ Kit for Sequencing CAT 450030 (according to manufacturer’s instructions).  Each ligation was transformed into Takara’s Stellar™ Competent <em>E. coli</em> HST08 strain cells (according to manufacturer’s instructions).  The resulting colonies obtained were PCR tested using M13 primer sets to confirm insertion.  Selected clones were Sanger sequenced using the M13 primers. BLAST analysis using the BLAST database (BLAST, 2023), on each of the sequenced amplicons was aligned to a region of the potato reference genome for comparison. Insertion sites for each event were analyzed for chromosomal deletions. Native Gene Interruption Analysis Determination of whether or not a native gene was interrupted by the insertion of the T-DNA in our potato events was studied. The potato reference genome used in this study is from the Potato Genome Sequencing Consortium and is a doubled monoploid (Diambra, L. A. 2011).  The sequence they obtained was integrated with a sequence from a heterozygous diploid line and used in the database potato reference genome <em>Solanum tuberosum</em> DM1-3 PGSC v4.04 pseudomolecules downloaded from http://spuddb.uga.edu/index.shtml. The insert location on each event was analyzed using the genome browser database within the sequence of the potato reference genome <em>Solanum tuberosum</em> DM1-3 PGSC v4.04 pseudomolecules located at http://spuddb.uga.edu/index.shtml to determine if there were any interruption of native genes. The database's graphical viewer was used to visually inspect the locus for disrupted genes.  Stability of T-DNA Across Clonal Cycles Potatoes are propagated asexually and both phenotypically and genetically identical to the mother plant as well as each other. The progeny plants (events) have not recombined meiotically.  The stability of the inserts in UB015, UB255, UG234, and UG265 events was assessed to show that DNA introduced into potato through transformation is stable over several clonal cycles. Genetic stability of the T-DNA inserts in UB015, UB255, UG234, and UG265 was assessed using PCR to determine the presence or absence of the insert in plants that have sustained three generations.  DNA insert stability was demonstrated in the originally transformed events (G0) by extracting and evaluating DNA from the events that had been propagated <em>in vitro.</em> For generation-3 (G3) analyses, 3 tubers from 3 plants from each event and a plant from each non-transgenic control were collected from an MSU confined field trial (East Lansing, MI) that sustained 3 tuber cycles (1 in greenhouse and 2 in field trials).  Genomic DNA from events UB015, UB255, UG234, and UG265 was isolated from fresh plant tissue using Qiagen DNeasy Plant Mini Kit Cat No./ID: 69104 according to the manufacturer’s instructions. PCR testing was done with the DNA primer sets found in the supplementary material (S2. Table 6). There are 2 primer sets located within the T-DNA. One set, called VNT1, is located in the native terminator of <em>Rpi-vnt1 </em>gene<em> </em>and the p4274 set is located in the promoter region of the <em>Rpi-mcq1</em>gene. The stability of the inserted T-DNA in the events is also shown by PCR analysis using primers that are unique to each event by using a primer set with one primer located in the T-DNA (at either the right or left border) and the other primer located in the chromosomal location specific to the event.  There are 4 sets of T-DNA right border region primer sets, one set specific for each event.  Each PCR was performed using conditions: 95°C 3 min, then 35 cycles at 95°C 30 sec, (Annealing Temp. see S2. Table 6)°C 30 sec, 72°C 1 min 20 sec.  The PCR samples were then electrophoresed on a 0.8% agarose gel using a 1 kb standard (STD) from (NEB 1 kb N3232S, New England Biolabs, USA).<strong> </strong>
description: This README.txt file was generated by Kelly Zarka **GENERAL INFORMATION**
 Title of Dataset: Dataset: T-DNA Characterization of Genetically Modified
 3-R-Gene Late Blight Resistant Potato Events with a Novel Procedure
 Utilizing the Samplix Xdrop® Enrichment Technology
 **Description of the data
 and file structure** **Plasmid Sequence**\ The plasmid pSIM4392 was
 developed and sequenced by Simplot Plant Sciences (Boise, ID). The genetic
 elements within the T-DNA are in the supplementary data (S2. Table 1).  A
 GenBanK file is provided and was used to analyze the integrity of the
 inserted T-DNA in each plant event described in the manuscript. **Plasmid
 files** pSIM4392_v2020_simplot.gbk: A GenBanK file of the pSIM4392
 plasmid                 pSIM4392_TDNA_LBtoRb.ape: A FASTA file of the
 T-DNA portion of the plasmid that  was transferred into each potato event
     **Inserted T-DNA analysis** Nanopore Genomic Sequencing and
 Bioinformatic Analysis\\\ Minion Oxford Nanopore Sequencing platform was
 used to generate long-read sequencing data from the dMDA samples as
 described by the manufacturer’s instructions (Premium whole genome
 amplification protocol (SQK-LSK109) with the Native Barcoding Expansions
 1-12 and 13-24 (EXP-NBD104/114)). Using the sequence data obtained and the
 potato reference genome *Solanum tuberosum* DM1-3 PGSC v4.04
 pseudomolecules downloaded from ,
 the T-DNA was mapped to the genome with Minimap2 and a bed file created
 corresponding to the mapping regions using bedtools bamtobed. After
 masking those regions using bedtools maskfasta, a new reference genome was
 created by adding the T-DNA only region of the pSIM4392 sequence as an
 extra chromosome.  All reads were then mapped to the new masked genome
 containing the T-DNA using Minimap2 with default settings for oxford
 nanopore reads (-ax map-ont).  The sequence viewer Integrative Genomics
 Viewer (IGV) was used to examine the coverage profile for the T-DNA. Both
 primary and supplementary mapping reads were extracted that map to the
 T-DNA using samtools view, specifying the reads to extract with the T-DNA
 coordinates (3), and seqkit grep. These are the reads of interest for
 finding insertions. Using the reference and the extracted reads from the
 T-DNA, Minimap2 was used to map the reads from the T-DNA to the masked
 genome which includes the T-DNA. Using samtools view a TAG was added
 containing the read name to the bam file. Then bedtools genomecov was used
 to generate of list of areas with coverage in the bam file. The IGV viewer
 was then used to identify the insertion borders. All sequences from each
 event were compared with the plasmid T-DNA to determine the integrity of
 the T-DNA sequences for each event. The bam files for each plant event are
 provided. **Inserted T-DNA files** MSU_DIA_UB015 bam files: *
 15const.reads.masked.genome.tag.bam.bai       
                                                          *
 UB015const.reads.masked.genome.tag.bam MSU_DIA_UB255 bam files: *
 UB255const.reads.masked.genome.tag.bam *
 UB255const.reads.masked.genome.tag.bam.bai MSU_GRA_UG234 bam files: *
 UG234const.reads.masked.genome.tag.bam *
 UG234const.reads.masked.genome.tag.bam.bai MSU_GRA_UG265 bam files: *
 UG265const.reads.masked.genome.tag.bam *
 UG265const.reads.masked.genome.tag.bam.bai **Flanking sequence analysis
 and Sanger Sequencing** The reads mapping to the construct were mapped
 back to the genome to identify reads that would span the breakpoint
 between insert and genome.  These sequences were then used to identify the
 chromosomal location of the inserts.  The identified insert site was
 confirmed by PCR across left and right breakpoints between insert and
 genome followed by Sanger sequencing. The PCR primers and conditions are
 found in S2. Table 4 and Table 5. The confirmation PCR analysis and Sanger
 Sequencing covered the junction as well as 1000bp of the flanking regions.
 Sanger sequencing files for each plant event are provided. **Flanking
 sequence analysis Sanger sequencing files** The folders below contain the
 Sanger sequences are .ab1 which contain the raw DNA sequence data as well
 as an .ape file that contains an annotated summary of the sequence data.
 ApE plasmid editor v2.0.61 was used to create the files. The program is
 available for download at
 UB015 Flanking
 Sanger Sequences.zip Folder: UB015 Left Border flanking sequence (contains
 Sanger sequence files that span the T-DNA junction and 1000bp of flanking
 chromosomal DNA at the left border of the T-DNA). Files: *
 KZ_1_A10_080_091120_7361_2020-09-11 chromosomal sequence at left border *
 KZ_1_UB15LB_ins_site_NPTII_pcr_2020-08-26 T-DNA junction sequence *
 KZ_2_UB15LB_NPTIIprimer_rev_2020-08-26 T-DNA junction sequence *
 UB15_Sanger_Seq_Left_border_summary Left border summary Folder: UB015
 Right Border flanking sequence (contains Sanger sequence files that span
 the T-DNA junction and 1000bp of flanking chromosomal DNA at the right
 border of the T-DNA) Files: * KZ_2_B10_078_091120_7361_2020-09-11 T-DNA
 junction sequence at right border *
 KZ_3_UB15RB_for1kb_UB15RBchr1_Fwd97_2020-09-11 chromosomal sequence at
 right border * KZ_4_UB15RB_for1kb_UB15REV831_2020-09-11 chromosomal
 sequence at left border * UB15_Sanger_Seq_Right_border_summary Right
 border summary UB255 Flanking Sanger Sequences.zip Folder: UB255 Left
 Border flanking sequence (contains Sanger sequence files that span the
 T-DNA junction and 1000bp of flanking chromosomal DNA at the left border
 of the T-DNA) Files: * KZ_1_UB255LB_Flank_2020-09-03_UB255LBprimer T-DNA
 junction sequence * KZ_2_UB255LB_Flank_2020-09-03 chromosomal sequence at
 left border * KZ_3_UB255LBextra_2020-09-03 chromosomal sequence at left
 border * KZ_4_UB255LBextra_GSPa_2020-09-03 chromosomal sequence at left
 border * UB255 Left Flanking Sequence summary Left border summary *
 UB255LB_Chr11_Flanking_seq_1kb_9_4_20seq extra Left border summary *
 UB255LB_Chr11_Flanking_seq_1kb_9_4_20seq_with_someTDNA_KZ2 extra Left
 border summary with additional T-DNA Folder: UB255 Right Border flanking
 sequence (contains Sanger sequence files that span the T-DNA junction and
 1000bp of flanking chromosomal DNA at the right border of the T-DNA)
 Files: * KZ_5_UB255RBchr11toborder_2020-08-26 T-DNA junction sequence at
 right border * KZ_6_UB255_rightborder_1kbflank_2020-08-26 chromosomal
 sequence at right border * UB255 Right Border Sequence summary Right
 border summary UG234 Flanking Sanger Sequences.zip Folder: UG234Left
 Border flanking sequence (contains Sanger sequence files that span the
 T-DNA junction and 1000bp of flanking chromosomal DNA at the left border
 of the T-DNA) Files: * KZ1_RAW_Seq_1_2021-01-05_UG234_Left_TDNA T-DNA
 junction and chromosomal sequence at left border *
 KZ1_RAW_Seq_1_2021-01-12_UG234_Left_TDNA T-DNA junction and chromosomal
 sequence at left border * UG234_Sanger_Left_border_seq_summary Left border
 summary Folder: UG234 Right Border flanking sequence (contains Sanger
 sequence files that span the T-DNA junction and 1000bp of flanking
 chromosomal DNA at the right border of the T-DNA) Files: *
 KZ2_RAW_Seq_1_2021-01-05_UG234_Right_TDNA T-DNA junction and chromosomal
 sequence at right border * KZ5_RAW_Seq_5_2021-01-05_UG234_Right_TDNA  
 T-DNA junction and chromosomal sequence at right border *
 KZ6_RAW_Seq_6_2021-01-05_UG234_Right_TDNA    T-DNA junction and
 chromosomal sequence at right border *
 UG234_Sanger_Right_border_seq_summary Right border summary
                                            UG265 Flanking Sanger
 Sequences.zip Folder: UG265Left Border flanking sequence (contains Sanger
 sequence files that span the T-DNA junction and 1000bp of flanking
 chromosomal DNA at the left border of the T-DNA) Files: *
 KZ_1_UG265_LeftBorderTDNA_2020-12-21 T-DNA junction and chromosomal
 sequence at left border * KZ_2_UG265_LeftFlank_1.8kb_2020-12-21
 chromosomal sequence at left border *
 KZ_7_UG265_LeftBorderTDNA_extra2020-12-21 T-DNA junction and chromosomal
 sequence at left border * UG265 Left Border Sequence summary Left border
 summary    Folder: UG265 Right Border flanking sequence (contains Sanger
 sequence files that span the T-DNA junction and 1000bp of flanking
 chromosomal DNA at the right border of the T-DNA) Files: *
 KZ_3_UG265_RightBorder_TDNA_2020-12-21 T-DNA junction and chromosomal
 sequence at right border * KZ_4_UG265_RightFlank1kb_2020-12-21 T-DNA
 junction and chromosomal sequence at right border *
 KZ_5_UG265_RightBorder_TDNA_extra_2020-12-21 extra T-DNA junction and
 chromosomal sequence at right border *
 KZ_6_UG265_RightFlank1kb2_extra_2020-12-21 extra T-DNA junction and
 chromosomal sequence at right border * UG265 Right Border Sequence summary
 Right border summary **Sharing/Access information** Links to other
 publicly accessible locations of the data: Solanum tuberosum DM1-3 PGSC
 v4.04 pseudomolecules:   NCBI
 BLAST database: 
 **Code/Software** | **Software/Tools/Resource** | **Link** | |
 :------------------------------------ |
 :------------------------------------------------ | | **bedtools** |
 | | **Guppy** |
 | | **Integrative
 Genomics Viewer (IGV)** | | |
 **minimap2** | | | **samtools** |
 | | **seqkit** |
 |