The Daffodil DNA Project: Results
This project is a collaboration between Jon Hale (Head of Biology at Beaulieu Convent School, Jersey), the University of Dundee School of Life Sciences and the James Hutton Institute.
Schools are working in conjunction with STEM partners from the University of Dundee and the James Hutton Institute to sample various daffodil varieties and carry out DNA sequencing of the chloroplast genome using Oxford Nanopore MinIon sequencing. The schools are then carrying out analysis of the resulting sequence to answer specific research questions.
The resulting sequencing data from all the schools are being collated on this site, allowing with additional sequences obtained by Beaulieu Convent School, Jersey. This enables the phylogeny of the various varietites to be inferred.
This project has been funded by The Royal Society, with additional funding from the Friends of Dundee University Botanic Garden to enable the University of Dundee Data Analysis Group to provide this centralised resource with consistent analysis processes.
We would be happy for anyone to reuse this data in their own projects, but please see our Data Usage Policy.
The sequences have all been processed using an automated workflow consisting of a number of stages allowing us to get from the raw data produced by the MinIon sequencer to assembled, annotated sequences
This is the process of converting the signal from the MinIon sequencer into the DNA bases this represents. The instrument produces thousands of reads of varying length, up to 40-50kb in length. Basecalling was carried out using the Oxford Nanopore Guppy software (version 6.1.2) using the appropriate high accuracy model for the flowcells and sequencing kits used (dna_r9.4.1_450bps_hac).
Every base of sequence generated is assigned a quality score, which is a measure of how likely the base is to have been called correctly. These range from 0-40, using a logarithmic scale, where a score of 10 represents a 1 in 10 chance that the base call is incorrect, a score of 20 relating to a 1 in 100 chance, and 30 being 1 in 1000. Sequences are filtered using NanoFilt to remove sequences shorter than 300 bp and also those with a minimum average quality score below 10.
A large proportion of these samples does not consist of daffodil chloroplastic DNA, so to make the assembly process easier, contaminants are removed from the sequence data. Kraken 2.1.2 is also used to identify what the origin of the seqeunces in each sample. The sequence reads are mapped to a known daffodil chloroplast sequence using winnowmap 2.03, and those which do not have similarity with this reference sequence are discarded.
- nextDenovo is a type of assembler known as a string-graph assembler. This requires longer reads, and is capable of producing a fully intact chloroplast assembly given sufficient data with long enough reads. nextDenovo is able to correctly assemble the inverted repeats, so is used first with each data set. If insufficient data is available, the assembly will fail.
- Flye is used if nextDenovo is unable to assemble the data. Flye is a repeat-graph assembler, which works in a different way to nextDenovo. There is typically far more data present in these samples than required for assembly, so the longest reads are used, with enough reads to cover the genome 50 times. Once assembled, the remaining reads are then mapped against the assembly to try to identify and remove errors in a process called polishing. 3 iterations of polishing were carried out in this case.
- Medaka is used first, which is a general polishing tool.
- Homopolish tries to correct a particular class of error which occurs when there is a run of the same base a number of times (known as homopolymer repeats). These can be a problem since it can be hard to determine quite how many times the base occurs, leading to expansion or compression of the repeat.
- The final tool (inspector) compares the assembly with the existing MH706763 assembly to try to identify any structural issues present.
2023 Daffodils
- Perianth: Yellow
- Corona: Yellow
- Division 2 (Large cupped daffodils)
- Hybridiser: P.D. Williams
- Registered: <1927
| Total Sequenced Bases (bp) | 329,228,066 | Mean Quality | 10.3 |
| Mean Read Length (bp) | 470.6 | N50 Read Length (bp) | 525.0 |
| Proportion of Chloroplast Bases (%) | 0.62 | Chloroplast Coverage | 12 |
| Assembled Contigs | 5 | Assembled Contig Length (bp) | 19047 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 120354 |
- Perianth: yellow
- Corona: yellow
- Division 7 (Jonquilla Daffodils)
- Hybridiser: Matthew Zandbergen
- Registered: 1948
| Total Sequenced Bases (bp) | 778,191,977 | Mean Quality | 10.2 |
| Mean Read Length (bp) | 3,036.5 | N50 Read Length (bp) | 5,395.0 |
| Proportion of Chloroplast Bases (%) | 4.5 | Chloroplast Coverage | 217 |
| Assembled Contigs | 1 | Assembled Contig Length (bp) | 184643 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 159064 |
- Perianth: white
- Corona: green/white/pink
- Pollen parent: Richardson
- Seed parent: May Queen
- Division 2 (Large cupped daffodils)
- Hybridiser: Brian Duncan
- Registered: 1979
| Total Sequenced Bases (bp) | 2,088,574,589 | Mean Quality | 10.6 |
| Mean Read Length (bp) | 1,455.0 | N50 Read Length (bp) | 2,173.0 |
| Proportion of Chloroplast Bases (%) | 2.3 | Chloroplast Coverage | 298 |
| Assembled Contigs | 3029 | Assembled Contig Length (bp) | 21321774 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 151524 |
- Perianth: white
- Corona: green/white/pink
- Pollen parent: Richardson
- Seed parent: May Queen
- Division 2 (Large cupped daffodils)
- Hybridiser: Brian Duncan
- Registered: 1979
| Total Sequenced Bases (bp) | 193,339,657 | Mean Quality | 10.4 |
| Mean Read Length (bp) | 2,434.5 | N50 Read Length (bp) | 4,564.0 |
| Proportion of Chloroplast Bases (%) | 2.5 | Chloroplast Coverage | 30 |
| Assembled Contigs | 7 | Assembled Contig Length (bp) | 117365 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 137260 |
Thanks to the Hardy Plant Society for the image.
- Perianth: white
- Corona: White with a green base and orange rim
- Pollen parent: Sylvia O'Neill
- Seed parent: Rubra
- Division 3 (Small cupped daffodils)
- Hybridiser: G.E.Mitsch
- Registered: 1959
| Total Sequenced Bases (bp) | 637,075,774 | Mean Quality | 10.4 |
| Mean Read Length (bp) | 974.3 | N50 Read Length (bp) | 1,280.0 |
| Proportion of Chloroplast Bases (%) | 2.8 | Chloroplast Coverage | 110 |
| Assembled Contigs | 21 | Assembled Contig Length (bp) | 157859 |
| Assembled Scaffolds | 2 | Assembled Scaffold Length (bp) | 159759 |
- Perianth: yellow
- Corona: orange
- Division 2 (Small cupped Daffodils)
- Hybridiser: Barr and Sons
- Registered: 1957
| Total Sequenced Bases (bp) | 853,626,090 | Mean Quality | 10.6 |
| Mean Read Length (bp) | 858.2 | N50 Read Length (bp) | 1,138.0 |
| Proportion of Chloroplast Bases (%) | 10 | Chloroplast Coverage | 534 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 172528 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 159291 |
- Perianth: yellow
- Corona: yellow
- Pollen parent: N. jonquilla
- Division 7 (Jonquilla Daffodils)
- Hybridiser: R.V. Favell
- Registered: <1939
| Total Sequenced Bases (bp) | 117,375,135 | Mean Quality | 10.1 |
| Mean Read Length (bp) | 2,650.9 | N50 Read Length (bp) | 4,438.0 |
| Proportion of Chloroplast Bases (%) | 5.4 | Chloroplast Coverage | 39 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 162909 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 159334 |
- Perianth: White
- Corona: yellow
- Division 1 (Trumpet daffodils)
- Hybridiser: J. Gerritsen & Son
- Registered: 1965
| Total Sequenced Bases (bp) | 329,245,778 | Mean Quality | 10.1 |
| Mean Read Length (bp) | 1,028.3 | N50 Read Length (bp) | 1,552.0 |
| Proportion of Chloroplast Bases (%) | 0.82 | Chloroplast Coverage | 16 |
| Assembled Contigs | 427 | Assembled Contig Length (bp) | 2918318 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 140215 |
- Perianth: White
- Corona: yellow
- Division 1 (Trumpet daffodils)
- Hybridiser: J. Gerritsen & Son
- Registered: 1965
| Total Sequenced Bases (bp) | 18,026,495 | Mean Quality | 10.2 |
| Mean Read Length (bp) | 423.2 | N50 Read Length (bp) | 466.0 |
| Proportion of Chloroplast Bases (%) | 0.62 | Chloroplast Coverage | 0 |
| Assembled Contigs | 0 | Assembled Contig Length (bp) | 0 |
| Assembled Scaffolds | 0 | Assembled Scaffold Length (bp) | 0 |
2022 Daffodils
- Perianth: white
- Division 3 (Small cupped Daffodils)
- Hybridiser: Rev G. H. Englehart
- Registered: <1891
| Total Sequenced Bases (bp) | 1,844,402,915 | Mean Quality | 10.5 |
| Mean Read Length (bp) | 2,408.6 | N50 Read Length (bp) | 3,782.0 |
| Proportion of Chloroplast Bases (%) | 2.8 | Chloroplast Coverage | 327 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 150611 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 150759 |
| Total Sequenced Bases (bp) | 148,406 | Mean Quality | 10.2 |
| Mean Read Length (bp) | 1,613.1 | N50 Read Length (bp) | 2,265.0 |
| Proportion of Chloroplast Bases (%) | 11 | Chloroplast Coverage | 0 |
| Assembled Contigs | 0 | Assembled Contig Length (bp) | 0 |
| Assembled Scaffolds | 0 | Assembled Scaffold Length (bp) | 0 |
| Total Sequenced Bases (bp) | 941,653 | Mean Quality | 10.1 |
| Mean Read Length (bp) | 973.8 | N50 Read Length (bp) | 1,556.0 |
| Proportion of Chloroplast Bases (%) | 11 | Chloroplast Coverage | 0 |
| Assembled Contigs | 0 | Assembled Contig Length (bp) | 0 |
| Assembled Scaffolds | 0 | Assembled Scaffold Length (bp) | 0 |
| Total Sequenced Bases (bp) | 689,139 | Mean Quality | 10.1 |
| Mean Read Length (bp) | 1,027.0 | N50 Read Length (bp) | 1,512.0 |
| Proportion of Chloroplast Bases (%) | 16 | Chloroplast Coverage | 0 |
| Assembled Contigs | 0 | Assembled Contig Length (bp) | 0 |
| Assembled Scaffolds | 0 | Assembled Scaffold Length (bp) | 0 |
- Corona: yellow
- Perianth: white
- Division: 1 (Trumpet Daffodils)
- Height: Standard (32-68cm)
- Hybridiser: William Backhouse
- Registered: <1869
| Total Sequenced Bases (bp) | 154,483,778 | Mean Quality | 11.3 |
| Mean Read Length (bp) | 1,906.5 | N50 Read Length (bp) | 3,539.0 |
| Proportion of Chloroplast Bases (%) | 17 | Chloroplast Coverage | 165 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 164568 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 165506 |
| Total Sequenced Bases (bp) | 16,703,346 | Mean Quality | 10.0 |
| Mean Read Length (bp) | 2,058.6 | N50 Read Length (bp) | 4,184.0 |
| Proportion of Chloroplast Bases (%) | 3.3 | Chloroplast Coverage | 3 |
| Assembled Contigs | 4 | Assembled Contig Length (bp) | 45827 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 131294 |
| Total Sequenced Bases (bp) | 660,329 | Mean Quality | 11.0 |
| Mean Read Length (bp) | 2,934.8 | N50 Read Length (bp) | 6,953.0 |
| Proportion of Chloroplast Bases (%) | 5 | Chloroplast Coverage | 0 |
| Assembled Contigs | 0 | Assembled Contig Length (bp) | 0 |
| Assembled Scaffolds | 0 | Assembled Scaffold Length (bp) | 0 |
- Corona: yellow
- Perianth: white
- Division: 2 (Large-Cupped Daffodils)
- Hybridiser: G.H. Engleheart
- Registered: <1898
| Total Sequenced Bases (bp) | 1,293,024,147 | Mean Quality | 12.1 |
| Mean Read Length (bp) | 1,072.8 | N50 Read Length (bp) | 1,558.0 |
| Proportion of Chloroplast Bases (%) | 37 | Chloroplast Coverage | 2,982 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 149075 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 153893 |
- Corona: yellow
- Perianth: white
- Division: 2 (Large-Cupped Daffodils)
- Hybridiser: G.H. Engleheart
- Registered: <1898
| Total Sequenced Bases (bp) | 43,051,165 | Mean Quality | 10.2 |
| Mean Read Length (bp) | 1,531.3 | N50 Read Length (bp) | 2,428.0 |
| Proportion of Chloroplast Bases (%) | 1.5 | Chloroplast Coverage | 3 |
| Assembled Contigs | 10 | Assembled Contig Length (bp) | 35625 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 128305 |
- Corona: yellow
- Perianth: white
- Division: 2 (Large-Cupped Daffodils)
- Hybridiser: The Brodie of Brodie
- Registered: <1911
| Total Sequenced Bases (bp) | 276,232,033 | Mean Quality | 11.9 |
| Mean Read Length (bp) | 751.6 | N50 Read Length (bp) | 1,024.0 |
| Proportion of Chloroplast Bases (%) | 55 | Chloroplast Coverage | 954 |
| Assembled Contigs | 10 | Assembled Contig Length (bp) | 184993 |
| Assembled Scaffolds | 2 | Assembled Scaffold Length (bp) | 208919 |
- Corona: orange
- Perianth: white
- Division: 3 (Small-Cupped Daffodils)
- Hybridiser: G.H. Engleheart
- Registered: <1897
| Total Sequenced Bases (bp) | 3,793,871,703 | Mean Quality | 10.7 |
| Mean Read Length (bp) | 2,344.6 | N50 Read Length (bp) | 4,325.0 |
| Proportion of Chloroplast Bases (%) | 1.9 | Chloroplast Coverage | 451 |
| Assembled Contigs | 1 | Assembled Contig Length (bp) | 161992 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 161992 |
- Corona: white
- Perianth: white
- Division: 3 (Small-Cupped Daffodils)
- Hybridiser: William Backhouse
- Registered: <1889
| Total Sequenced Bases (bp) | 637,388,752 | Mean Quality | 10.3 |
| Mean Read Length (bp) | 1,562.3 | N50 Read Length (bp) | 2,449.0 |
| Proportion of Chloroplast Bases (%) | 1.7 | Chloroplast Coverage | 65 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 127041 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 143789 |
| Total Sequenced Bases (bp) | 11,975,706 | Mean Quality | 10.4 |
| Mean Read Length (bp) | 1,217.8 | N50 Read Length (bp) | 2,056.0 |
| Proportion of Chloroplast Bases (%) | 9 | Chloroplast Coverage | 6 |
| Assembled Contigs | 12 | Assembled Contig Length (bp) | 70605 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 127240 |
- Perianth: white
- Division 9 (Poeticus Daffodils)
- Selected: James Walker
- Registered: <1870
| Total Sequenced Bases (bp) | 172,644,669 | Mean Quality | 10.3 |
| Mean Read Length (bp) | 2,731.9 | N50 Read Length (bp) | 4,175.0 |
| Proportion of Chloroplast Bases (%) | 9.9 | Chloroplast Coverage | 106 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 143892 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 149951 |
| Total Sequenced Bases (bp) | 13,958,953 | Mean Quality | 10.3 |
| Mean Read Length (bp) | 2,510.2 | N50 Read Length (bp) | 5,812.0 |
| Proportion of Chloroplast Bases (%) | 6.9 | Chloroplast Coverage | 6 |
| Assembled Contigs | 6 | Assembled Contig Length (bp) | 105128 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 130314 |
- Perianth: white
- Division 13 - Species, Wild Variants
| Total Sequenced Bases (bp) | 4,063,677,562 | Mean Quality | 10.2 |
| Mean Read Length (bp) | 3,396.6 | N50 Read Length (bp) | 5,846.0 |
| Proportion of Chloroplast Bases (%) | 1.4 | Chloroplast Coverage | 353 |
| Assembled Contigs | 2 | Assembled Contig Length (bp) | 159765 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 159865 |
| Total Sequenced Bases (bp) | 4,591,656 | Mean Quality | 10.1 |
| Mean Read Length (bp) | 912.5 | N50 Read Length (bp) | 1,442.0 |
| Proportion of Chloroplast Bases (%) | 12 | Chloroplast Coverage | 3 |
| Assembled Contigs | 6 | Assembled Contig Length (bp) | 43778 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 142227 |
- Perianth: white
- Division 1 (Trumpet Daffodils)
- Hybridiser: Unknown
- Registered: <1830
| Total Sequenced Bases (bp) | 558,705,420 | Mean Quality | 10.2 |
| Mean Read Length (bp) | 740.5 | N50 Read Length (bp) | 1,020.0 |
| Proportion of Chloroplast Bases (%) | 0.13 | Chloroplast Coverage | 4 |
| Assembled Contigs | 6 | Assembled Contig Length (bp) | 44844 |
| Assembled Scaffolds | 1 | Assembled Scaffold Length (bp) | 72568 |
| Total Sequenced Bases (bp) | 4,398,963 | Mean Quality | 10.0 |
| Mean Read Length (bp) | 910.8 | N50 Read Length (bp) | 1,295.0 |
| Proportion of Chloroplast Bases (%) | 5.1 | Chloroplast Coverage | 1 |
| Assembled Contigs | 0 | Assembled Contig Length (bp) | 0 |
| Assembled Scaffolds | 0 | Assembled Scaffold Length (bp) | 0 |
Genomic Multiple Sequence Alignments
Two multiple sequence alignments have been carried out. The first includes all (including partial) sequences, while the second includes just sequences > 120kb in length, which were used for carrying out the phylogenetic analysis.
The JalviewJS link below for the 'long' sequences will also load they phylogenetic tree in addition to the alignment.
Phylogeny
Protein Multiple Sequence Alignments
Each identified gene within the assembled chloroplast genomes has been translated to a protein sequence, and then a multiple sequence alignment generated for each gene. Alignments were created using Muscle 5.1 with default parameters.