Tutorial

Tutorial: plastomes of 28 potato cultivars

This tutorial runs BACoN on public data: whole-genome Nanopore reads of 28 potato (Solanum tuberosum) cultivars from the Ural region, sequenced to compare their plastomes (NCBI BioProject PRJNA807056; MinION, reads longer than 6 kb, 100–700 Mb per sample). Potato cultivars carry a few types of plastome ("cytoplasm types"); T-type plastomes carry a 241 bp deletion between the ndhC and trnV-UAC genes that the other types lack (Kawagoe & Kikuta 1991; Hosaka 2002). Let's see what BACoN finds.

1. Download the reads and the reference

About 7 GB. The script downloads the 28 runs from ENA, named after their cultivar, and checks their MD5:

mkdir potato && cd potato
curl -s "https://www.ebi.ac.uk/ena/portal/api/filereport?accession=PRJNA807056&result=read_run&fields=sample_alias,fastq_ftp,fastq_md5&format=tsv" \
    | tail -n +2 > runs.tsv
mkdir -p reads
while IFS=$'\t' read -r run alias url md5; do
    name=$(echo "$alias" | tr ' ' '_')
    curl -s -C - --retry 5 -o "reads/$name.fastq.gz" "https://$url"
    echo "$md5  reads/$name.fastq.gz"
done < runs.tsv > md5.txt
md5sum -c md5.txt

# The reference: the plastome of cultivar Désirée
curl -s "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=NC_008096.2&rettype=fasta" \
    > NC_008096.2.fasta

If an MD5 check fails, run the loop again: curl -C - resumes interrupted downloads.

2. Run BACoN

bacon -r NC_008096.2.fasta -i reads/ -o bacon_potato -t 32 -p 8

The defaults: baiting with minimap2, Filtlong capping each sample at 100x, templated assembly with samtools, core SNPs with SKA2, and a FastTree tree. It takes about 3 minutes.

Open bacon_potato/report.html in a browser for everything below on one page. The report of this run is online: view it or download it (local paths removed). The next sections look at the files behind it.

3. Reads and assemblies: summary.tsv

Sample     Status  Raw_reads  Raw_bases  Baited_reads  Baited_pct  Filtered_reads  Est_depth  Assembly_length  N_bases
12_22_134  ok      18388      202618009  3553          17.437      1433            100.0      155386           133
14_4_1     ok      15023      182354649  3018          18.349      1313            100.1      155383           153
14_6_3     ok      20396      234625686  4122          17.970      1292            100.2      155166           0
...

• 10–23% of the bases are plastid reads, typical of leaf DNA; three runs (Alaska, Argo, Shah) were already filtered to plastid reads (nearly 100%).
• Every sample reaches 64–100x after filtering, far more than needed.
• The consensus lengths fall into two groups: about 155,170 bp and about 155,390 bp. Samples of the second group have 119–153 N bases, nearly all in one region, next to position 52,580.

4. SNPs and tree: 4_compared/ska/

snp_distances.tsv gives 119 SNP sites between the 28 plastomes and the reference. The cultivars fall into three groups:

Group	Cultivars	SNPs to the reference	Within the group
T-type	14_6_3, 16-35-5, 16_1_2, Bagira, Bankir, Iskra, Luks, Shah, Terra, Zdraven	1	identical
Lineage A	15-27-1, Legenda	67	identical
Lineage B	12_22_134, 14_4_1, 15_22_4, 16_4_3, Alaska, Amur, Argo, Baron, Bravo, Gornyak, Irbitskiy, Kamenskiy, Mishka, Otrada, Start, Utro_ranneye	67–76	0–13

Lineage A is 66 SNPs from the T-type group, lineage B 66–75, and the two lineages are 76–85 SNPs apart. Lineage B holds three distinct plastomes: 11 identical cultivars, a group of four (14_4_1, 16_4_3, Baron, Start) 5 SNPs away, and 15_22_4, 12–13 SNPs from both.

The T-type group shares the reference's plastome but for one SNP. Lineages A and B are not T-type (next section); which of the other cytoplasm types they are is not determined here.

5. The 241 bp marker

The N bases of lineages A and B sit in the ndhC–trnV-UAC spacer (reference positions 51,834 to 52,696). Aligning an assembly of each group to the reference shows why:

minimap2 -cx asm5 --cs NC_008096.2.fasta bacon_potato/3_assembled/all_assemblies/Alaska.fasta

the plastomes of lineages A and B have an insertion of about 240 bp at position 52,578 relative to the T-type reference: the known T-type deletion, seen from the other side. The templated assembly places it, but the reads do not agree on all of its bases, hence the N. A de novo assembly resolves it completely:

bacon -r NC_008096.2.fasta -i reads/ -o bacon_potato -a flye -t 48 -p 12

reuses the baited and filtered reads and assembles each sample with Flye (about 10 minutes). The Flye assembly of Alaska has an insertion of exactly 241 bp at position 52,578. Flye also reports 17 of the 28 plastomes as one circular contig. Its SNP distances (4_compared/ska/snp_distances.tsv, replaced) are identical to those of the templated assembly for all 406 pairs of genomes (the 28 cultivars and the reference).

What this shows

• Genome skimming data from a few hundred megabases per sample is enough for complete plastomes.
• The templated assembly gives the SNPs and small indels in minutes; a de novo assembly resolves the insertions and the structure, and confirms the SNPs independently.
• Identical plastomes (distance 0) are common: these 28 cultivars carry only five distinct plastomes.

References

• Kawagoe Y., Kikuta Y. (1991) Chloroplast DNA evolution in potato (Solanum tuberosum L.). Theoretical and Applied Genetics 81:13–20. https://doi.org/10.1007/BF00226106
• Hosaka K. (2002) Distribution of the 241 bp deletion of chloroplast DNA in wild potato species. American Journal of Potato Research 79:119–123. https://doi.org/10.1007/BF02881520