TITLE: Vaccinium floribundum Genome Assembly and Annotation Script
AUTHORS: Martina Albuja-Quintana, Gabriela Pozo, Milton Gordillo-Romero, Carolina E. Armijos, Maria de Lourdes Torres

[DESCRIPTION]
Oxford Nanopore long reads and Illumina short reads obtained from sequencing the DNA of a Vaccinum floribundum specimen from the Paramo region in Ecuador, were used to assemble and annotate the whole genome of this species. ONT long reads were filtered and trimmed in Nanofilt and Porechop. Sequencing statistics were visualized in Nanoplot. Illumina short reads were evaluated with fastqc. Different assemblers and polishers were used with both long and short reads. The resulting Flye assembly polished with Polca and Medaka was then analyzed for genome completeness and quality with Quast, BUSCO, LAI Index, and Coverage Graph. The assembly was later annotated in Maker in 3 consecutive rounds using the ab initio gene predictor SNAP.

[STEPS]
SECTION: Oxford Nanopore Sequencing - Raw Reads Filtering, Trimming, and Statistics
1. Raw Read Adapter Filtering 
Porechop v0.2.4 (RRID:SCR_016967)

 

SECTION: Oxford Nanopore Sequencing - Raw Reads Filtering, Trimming, and Statistics
2. Raw Read Quality and Length Trimming
Nanofilt v2.8.0 (RRID:SCR_016966)

 

SECTION: Oxford Nanopore Sequencing - Raw Reads Filtering, Trimming, and Statistics
3. Raw Read Dataset Statistics
LongQC v1.2.0c

 

Nanoplot v1.33.0 (RRID:SCR_024128)

 

SECTION: Illumina Sequencing - Raw Reads Statistics
4. Raw Read Dataset Statistics
FastQC (RRID:SCR_014583) 

 

SECTION: Genome Size Estimation
5. k-mer based analysis
Jellyfish v2.3.0 (RRID:SCR_005491)

 

k-mer profile visualization
GenomeScope v2.0 (RRID:SCR_017014)

 

SECTION: De novo Genome Assembly and Polishing
6. Assembly 
SMARTdenovo v1.0.0 (RRID:SCR_017622)

 

Flye v2.9.2 (RRID:SCR_017016) 

 

MaSuRCA v.4.1.0 (RRID:SCR_010691)

 

SECTION: De novo Genome Assembly and Polishing
7. Polishing
Medaka v1.11.1 

 

POLCA (MaSuRCA, v4.1.0 RRID:SCR_010691) 

 

SECTION: Genome assembly quality, continuity, and completeness assessment
8. Quast v5.2.0 (RRID:SCR_001228)

 

SECTION: Genome scaffolding
12. ntLink v1.3.9 

 

SECTION: Removing Foreign Contamination
13. python3 ./fcs.py screen Assembly.fasta --fasta ./fcsgx_test.fa.gz --out-dir ./gx_out/ --gx-db "$GXDB_LOC/gxdb" --tax-id 6973
SECTION: Genome Annotation
14. RepeatModeler v2.0.3 (RRID:SCR_015027)

 

SECTION: Genome assembly quality, continuity, and completeness assessment
9. BUSCO v5.4.7 (RRID:SCR_015008)

 

SECTION: Genome assembly quality, continuity, and completeness assessment
11. Coverage Graphs of ONT and Illumina Reads

1. Change contig names from assembly file

 

2. Identify longest contig

 

3. Extract the longest contig from the assembly
Samtools package v1.18 (RRID:SCR_002105)

 

4. Extract 10% of ONT and Illumina reads (total reads were obtained from previously run Nanoplot)
BWA (RRID:SCR_010910)

 

5. Map ONT reads and illumina reads, seperately, to the assembly

ONT reads: minimap2 v2.26 (RRID:SCR_018550) 

 

Illumina reads: BWA (RRID:SCR_010910)

 

6. Convert sam files to bam

 

7. Sort bam files 

 


8. Coverage depth
Samtools package v1.18 (RRID:SCR_002105)

 

5. Graphs: script developed by López et al., 2023

 

SECTION: Genome assembly quality, continuity, and completeness assessment
10. Long Terminal Repeat (LTR) Assembly Index (LAI) 

LTRharvest v1.6.2 (RRID:SCR_018970)

1. Create an "Enhanced Suffix Array"

 

2. Run LTRHARVEST

 


LTR_FINDER v1.0.7 (RRID:SCR_015247)

 

Combine both documents from LTRFinder and LTRHarvest

 


LTR_retriever v2.8.7 (RRID:SCR_017623)

 

SECTION: Genome Annotation
16. Generate Executable Files

 

SECTION: Genome Annotation
15. Change Contig Names in Assembly File

 

SECTION: Genome Annotation
17. 1. Modify Maker_opts.ctl file

2. Run 10 iterations of the first run of maker

 

3. Run and train SNAP script

 

4. Run 5 iterations of the second run of maker

 

5. Rerun and train SNAP script

 

4. Run 5 iterations of the third run of maker

 

SECTION: Genome Annotation
18. Generating and Filtering Final Files

1. Generate single gff and fasta protein and transcript files from all 3 maker rounds

 

2. Identify conserved protein regions in predicted gene models 
InterProScan v5.64-9.60 (RRID:SCR_005829)

 

3. Modify the original gff3 file by identifying gene models with conserved protein domains

 

4. Eliminate gene models with AED <1

 

5. Calculate annotation statistics with AGAT
AGAT (Another Gff Analysis Toolkit) v1.2.0 

 

6. Filter out gene models with no conserved protein regions and AED <1 from protein and transcript fasta files