Prunus fruticosa 27e12(2) Whole Genome v1.0 Assembly & Annotation

Overview
Analysis NamePrunus fruticosa 27e12(2) Whole Genome v1.0 Assembly & Annotation
MethodAssembler: Canu, Polishing: Pilon, Annotation: MAKER, Manual Annotation: Apollo
SourcePacBio long-reads & Illumina short-reads (MSU)
Date performed2023-01-10

Publication:

Goeckeritz, C. Z., Rhoades, K. B., Childs, K., Iezzoni, A. F., VanBuren, R., Hollender, C. A. The genome of the segmental allotetraploid sour cherry (Prunus cerasus) for genomics-assisted breeding. Submitted to Genome Biology

A chromosome-scale assembly for tetraploid sour cherry (Prunus cerasus L.) ‘Montmorency’ identifies three distinct ancestral Prunus genomes
Charity Z. Goeckeritz, Kathleen E. Rhoades, Kevin L. Childs,  Amy F. Iezzoni, Robert VanBuren, Courtney A. Hollender
doi: https://doi.org/10.1101/2023.01.10.523438

Overview

Three collections of young leaf tissue were made in the spring of 2019 for PacBio sequencing, Illumina sequencing of a gDNA library, and Illumina sequencing of a Hi-C library. Canu was used to assemble approximately 100X PacBio long-reads into contigs. Subsequently, 56X coverage of Illumina 150bp paired-end gDNA reads were aligned to the assembly using bowtie2, and these alignments were used for polishing the initial assembly with Pilon. After polishing, the assembly was scaffolded using Illumina 150bp paired-end Hi-C reads and the Juicer + 3D-DNA pipeline. Misassemblies in the scaffolded chromosome-scale sequences were corrected in JuiceBox Assembly Tools. For annotation, Illumina 150bp paired-end RNA-sequencing was conducted on a variety of tissues from cultivar 'Montmorency' at different developmental time points to aid in gene discovery. In addition to the RNA-seq, Nanopore cDNA-sequencing was conducted on a diverse pool of tissues in an attempt to obtain full-length transcripts for annotation. Manually-curated protein databases were downloaded from Uniprot and arabidopsis.org and aligned to the assembly with Exonerate to make homology-based gene predictions. The RNA-seq and cDNA-sequencing data were aligned to the assembly using STAR and minimap2, respectively, and transcriptomes from both types of data were created using Stringtie. The two transcriptomes and protein alignments were given to MAKER for evidence-based gene predictions. After MAKER's first run, the resulting .gff3 was used to train gene finders Augustus and SNAP. MAKER was run a second time using these evidence-trained gene finders and the subsequent gene predictions were filtered so that each prediction contained at least one known Pfam domain. Then, deFusion was run to identify erroneously fused gene candidates, and more than 2500 gene predictions on the 16 main chromosomes of the assembly were defused as a result of this pipeline. At this point, predictions were again filtered to contain Pfam domains but also to exclude predictions with homology to transposeable elements. Lastly, Apollo was used to manually annotate the intron-exon structure of 67 genes, including all identified DAM (Dormancy Associated MADS-box) genes.

Summary of the ‘Montmorency’ assembly metrics

Estimated haploid genome size (kmer analysis; k = 25)

621 Mb

Estimated Heterozygosity (total)

4.90%

class

aaab  

aabb

aabc

abcd

 

2.430%

2.060%

0.001%

0.451%

Full assembly size

1066 Mb

Scaffolded assembly size

771.8 Mb

NG50

11.56 Mb

Number of contigs

3592

Linkage Groups

24

BUSCO (Viridiplantae db10)

complete

singletons

duplicates

missing

fragmented

Scaffolded assembly (24 LGs)

98.6%

5.4%

93.2%

0.9%

0.5%

subgenome A

91.5%

89.6%

1.9%

6.4%

2.1%

subgenome A'

94.8%

92.9%

1.9%

4.0%

1.2%

subgenome B

90.8%

88.2%

2.6%

7.3%

1.9%

Estimated % Repeats (Full Assembly)

LTR

TIR

Helitron

Total

35.6%

11.6%

1.3%

48.5%

LTR Assembly Index

Full assembly (incl. unanchored)

14.74

Scaffolded assembly

17.09

 

 

Homology

Homology of the Prunus fruticosa 27e12(2) Genome v1.0 proteins was determined by pairwise sequence comparison using the blastp algorithm against various protein databases. An expectation value cutoff less than 1e-9 was used for the NCBI nr (Release 2021-09) and 1e-6  for the Arabidoposis proteins (Araport11), UniProtKB/SwissProt (Release 2021-09), and UniProtKB/TrEMBL (Release 2021-09) databases. The best hit reports are available for download in Excel format. 

 

Protein Homologs

Prunus fruticosa v1.0 proteins with NCBI nr homologs (EXCEL file) pfruticosa_v1.0_vs_nr.xlsx.gz
Prunus fruticosa v1.0 proteins with NCBI nr (FASTA file) pfruticosa_v1.0_vs_nr_hit.fasta.gz
Prunus fruticosa v1.0 proteins without NCBI nr (FASTA file) pfruticosa_v1.0_vs_nr_noHit.fasta.gz
Prunus fruticosa v1.0 proteins with arabidopsis (Araport11) homologs (EXCEL file) pfruticosa_v1.0_vs_arabidopsis.xlsx.gz
Prunus fruticosa v1.0 proteins with arabidopsis (Araport11) (FASTA file) pfruticosa_v1.0_vs_arabidopsis_hit.fasta.gz
Prunus fruticosa v1.0 proteins without arabidopsis (Araport11) (FASTA file) pfruticosa_v1.0_vs_arabidopsis_noHit.fasta.gz
Prunus fruticosa v1.0 proteins with SwissProt homologs (EXCEL file) pfruticosa_v1.0_vs_swissprot.xlsx.gz
Prunus fruticosa v1.0 proteins with SwissProt (FASTA file) pfruticosa_v1.0_vs_swissprot_hit.fasta.gz
Prunus fruticosa v1.0 proteins without SwissProt (FASTA file) pfruticosa_v1.0_vs_swissprot_noHit.fasta.gz
Prunus fruticosa v1.0 proteins with TrEMBL homologs (EXCEL file) pfruticosa_v1.0_vs_trembl.xlsx.gz
Prunus fruticosa v1.0 proteins with TrEMBL (FASTA file) pfruticosa_v1.0_vs_trembl_hit.fasta.gz
Prunus fruticosa v1.0 proteins without TrEMBL (FASTA file) pfruticosa_v1.0_vs_trembl_noHit.fasta.gz

 

Assembly

The Prunus fruticosa 27e12(2) Genome v1.0 assembly file is available in FASTA format.

Downloads

Contigs (FASTA file) pfruticosa_v1.0.fasta.gz
Contigs hardmasked(FASTA file) pfruticosa_v1.0.hardmasked.fasta.gz

 

Gene Predictions

The Prunus fruticosa 27e12(2) v1.0 genome gene prediction files are available in FASTA and GFF3 formats.

Downloads

Protein sequences  (FASTA file) pfruticosa_v1.0.proteins.fasta.gz
CDS  (FASTA file) pfruticosa_v1.0.cds.fasta.gz
Transcript sequences  (FASTA file) pfruticosa_v1.0.transcripts.fasta.gz
Gene sequences  (FASTA file) pfruticosa_v1.0.genes.fasta.gz
Genes (GFF3 file) pfruticosa_v1.0.genes.gff3.gz

 

Functional Analysis

Functional annotation for the Prunus fruticosa 27e12(2) Genome v1.0 are available for download below. The Prunus fruticosa 27e12(2) Genome v1.0 proteins were analyzed using InterProScan in order to assign InterPro domains and Gene Ontology (GO) terms. Pathways analysis was performed using the KEGG Automatic Annotation Server (KAAS).

Downloads

GO assignments from InterProScan pfruticosa_v1.0_genes2GO.xlsx.gz
IPR assignments from InterProScan pfruticosa_v1.0_genes2IPR.xlsx.gz
Proteins mapped to KEGG Orthologs pfruticosa_v1.0_KEGG-orthologis.xlsx.gz
Proteins mapped to KEGG Pathways pfruticosa_v1.0_KEGG-pathways.xlsx.gz

 

Transcript Alignments
Transcript alignments were performed by the GDR Team of Main Bioinformatics Lab at WSU. The alignment tool 'BLAT' was used to map transcripts to the Prunus fruticosa27e12(2) genome assembly. Alignments with an alignment length of 97% and 97% identify were preserved. The available files are in GFF3 format.

 

Fragaria x ananassa GDR RefTrans v1 pfruticosa_v1.0_f.x.ananassa_GDR_reftransV1
fragaria avium GDR RefTrans v1 pfruticosa_v1.0_p.avium_GDR_reftransV1
fragaria persica GDR RefTrans v1 pfruticosa_v1.0_p.persica_GDR_reftransV1
Rosa GDR RefTrans v1 pfruticosa_v1.0_rosa_GDR_reftransV1
Rubus GDR RefTrans v2 pfruticosa_v1.0_rubus_GDR_reftransV2
Malus_x_domestica GDR RefTrans v1 pfruticosa_v1.0_m.x.domestica_GDR_reftransV1
Pyrus GDR RefTrans v1 pfruticosa_v1.0_pyrus_GDR_reftransV1