Dataset DOI: 10.5061/dryad.rjdfn2zsm

Description of the data and file structure

Plasmid Sequence
The plasmid construct pINVBP1 was used for an Agrobacterium meditated potato transformation (Bhaskar, P.B., 2010). Information on all of the genetic elements in plasmid pINVBP1 can be found in the Supplemental Table S.1.

The sequence of the transformation plasmid pINVBP1 is provided. Additionally, a separate file containing only the T-DNA (the portion of the plasmid that was transferred to the Kal91.3 potato event).\

Inserted T-DNA analysis

The Kal91.3 T-DNA sequence data was generated by first utilizing Xdrop™ enrichment technology and second by Sanger sequence technology. Utilization of the Xdrop™ technology for characterization of T-DNA can be reviewed in our recent publication (Zarka, KA. et al., 2024). Kal91.3 bioinformatic analysis is as follows. To obtain sequences with high accuracy, the generated raw data files were base-called using Guppy 5.0.17 with super high accuracy and quality 10 filtering settings. Using the sequence data and the potato reference genome Solanum tuberosum 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. After masking those regions, a new reference genome was created by adding the T-DNA only region of the pINVBP1 sequence as an extra chromosome. All reads were then mapped to the new masked genome containing the T-DNA. 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 and these are the reads of interest for finding insertions. Using the reference and the extracted reads from the T-DNA, a list of areas was generated with coverage in the bam file. The IGV viewer was then used to identify the insertion borders. The bioinformatic analysis files are provided.

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 insert and in the case of Kal91.3 there are two inserts. The identified insert sites were confirmed by PCR across left and right breakpoints between insert and genome followed by Sanger sequencing. The PCR primers and conditions are found in Supplementary Table S2. The confirmation PCR analysis and Sanger Sequencing covered the junction as well as 1000bp of the flanking regions. Sanger sequencing files for each insert are provided.

The folders contain the Sanger sequences ( .ab1 files), 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 https://jorgensen.biology.utah.edu/wayned/ape/ and has been published.

Davis MW and Jorgensen EM (2022) ApE, A Plasmid Editor: A Freely Available DNA Manipulation and Visualization Program. Front. Bioinform. 2:818619.

Files and variables

Plasmid files

• Zarka_AG_1_KZ_pINVBP1.fasta: A FASTA file of the pINVBP1 plasmid

• Zarka_AG_1_KZ_pINVBP1_TDNA_ONLY.ape: A FASTA file of the T-DNA portion of the plasmid that was transferred into each potato event

Inserted T-DNA files

Kal91.3 bioinformatic analysis files:

• const.reads.masked.genome.tag.bam.bai
• const.reads.masked.genome.tag.bam
• const.reads.masked.genome.tag.bam.fai
• ref_tdna_masked
• ref_tdna_masked.fasta.fai

Flanking Sequence Analysis files:

File:

• Flanking_Analysis_Sanger_Raw_Data.zip

Folder: Flanking Analysis Sanger Raw Data

Subfolder: Kal91_3 Chr1 LB Raw Seq

Files:

• Sanger_Kal91_chr1_LB_Chr_98bp_end_PCR_2_D

• Sanger_Kal91_chr1_LB_Chr_299bp_internal_PCR_2_primerE

• Sanger_Kal91_chr1_LB_Chr_603bp_from border_PCR_1_C

• Sanger_Kal91_chr1_LB_TDNA_PCR_1_B

Subfolder: Kal91_3 Chr1 RB Raw Seq

Files:

• Sanger_PCR3_G_Kal91_chr1_RB_TDNAjunct_96bp

• Sanger_PCR4_H_Kal91_chr1_RB_chr_inter_262bp

• Sanger_PCR4_I_Kal91_chr1_RB_chr_642bp to end

Subfolder: Kal91_3 Chr3 LB Raw Seq

Files:

• Sanger_J_Kal91_chr3_LB_chr int_510bp

• Sanger_L_Kal91_chr3_LB_chr end_34bp

• Sanger_M1_Kal91_chr3_LB__500bpTDNA_83bp_chr

• Sanger_M2_Kal91_chr3_LB_chr int_373bp

Subfolder: Kal91_3 Chr3 RB Raw Seq

Files:

• Sanger_Kal91_chr3_RB_Chr_740bp_PCR_6_P

• Sanger_Kal91_chr3_RB_TDNA_PCR_6_N

Folder: Flanking_Analysis_APE_Annotated.zip

Files:

• PCR1_Primer 1_B_Kal91.3 chr 1 Left T-DNA sanger

• PCR1_Primer C_Kal91.3 chr 1 Left Chr at junction 603bp sanger

• PCR2_Primer 2_D_Kal91.3 chr 1 Left chr End 98bp Sanger

• PCR2_Primer 2_E_Kal91.3 chr 1 Left chr Internal 307bp sanger

• PCR3_Primer 3_G_Kal91.3 chr1 Right 500bp T_DNA_ 96bp chr Sanger

• PCR4_Primer 4_H_Kal91.3 chr1 Right chromosomal internal 262bp Sanger

• PCR4_Primer 4_I_Kal91.3 chr1 Right chromosomal End 642bp Sanger

• PCR5_ Primer 5_L_Kal91.3 chr 3 Left chromosomal seq end 34p Sanger

• PCR5_ Primer J_Kal91.3 chr 3 Left chr sanger seq near junction 510bp

• PCR5_ Primer M1_Kal91.3 chr 3 Left 500bp of T_DNA and 83bp Junction seq

• PCR5_ Primer M2_Kal91.3 chr 3 Left chromosomal sanger seq 373bp

• PCR6_Primer_6_N_Kal91.3 chr 3 Right T-DNA 500 plus 360 chr Sanger

• PCR6_Primer_6_P_Kal91.3 chr 3 Right chromosomal 740bp Sanger

Code/software

Access information

Other publicly accessible locations of the data:

• Solanum tuberosum DM1-3 PGSC v4.04 pseudomolecules: http://spuddb.uga.edu/index.shtml
• NCBI BLAST database: https://blast.ncbi.nlm.nih.gov/Blast.cgi