III. Assembling plastid genomes - barrettlab/2021-Genomics-bootcamp GitHub Wiki
Plastid genome assembly with Fast-Plast, NOVOplasty, and GetOrganelle
Links to GitHub pages:
1. Seed and assemble: de novo plastome assembly with NOVOplasty
NOVOplasty is a perl script that requires three files (or more, optionally):
i. Your reads (either R1.fastq + R2.fastq, or a "shuffled" R1R2.fastq file) ii. A "seed," for example a closely related rbcL sequence from Genbank iii. A configuration file. This is just a manually edited text file pointing to your reads, the seed, etc., and supplying some parameters.
Here is an example config file (e.g. config.txt)
Project:
-----------------------
Project name = Mitochondrial_genome_assembly_01
Type = mito
Genome Range = 12000-20500
K-mer = 39
Max memory = 11
Extended log =
Save assembled reads =
Seed Input = seed.fasta
Reference sequence =
Variance detection =
Chloroplast sequence =
Dataset 1:
-----------------------
Read Length = 151
Insert size = 412
Platform = illumina
Single/Paired = PE
Combined reads =
Forward reads = R1.fq.gz
Reverse reads = R2.fq.gz
Heteroplasmy:
-----------------------
MAF =
HP exclude list =
PCR-free =
Optional:
-----------------------
Insert size auto = yes
Insert Range = 1.9
Insert Range strict = 1.3
Use Quality Scores = no
Project:
-----------------------
Project name = Hexalectris_warnockii_plastome
Type = chloro
Genome Range = 50000-160000
K-mer = 41
Max memory =
Extended log =
Save assembled reads =
Seed Input = rps14_seed.fasta
Reference sequence =
Variance detection =
Chloroplast sequence =
Dataset 1:
-----------------------
Read Length = 101
Insert size = 412
Platform = illumina
Single/Paired = PE
Combined reads =
Forward reads = Hexalectris_warnockii_R1.fq.gz
Reverse reads = Hexalectris_warnockii_R2.fq.gz
Heteroplasmy:
-----------------------
MAF =
HP exclude list =
PCR-free =
Optional:
-----------------------
Insert size auto = yes
Insert Range = 1.9
Insert Range strict = 1.3
Use Quality Scores = no
Project1
/path/to/seed_file/Seed1.fasta
/path/to/reads/reads_1a.fastq
/path/to/reads/reads_2a.fastq
Project2
/path/to/seed_file/Seed2.fasta
/path/to/reads/reads_1b.fastq
/path/to/reads/reads_2b.fastq
Project3
/path/to/seed_file/Seed3.fasta
/path/to/reads/reads_1c.fastq
/path/to/reads/reads_2c.fastq
# etc...
Project:
-----------------------
Project name = batch:/path/to/batch_file.txt
Type = mito
Genome Range = 12000-20500
K-mer = 39
Max memory = 11
Extended log =
Save assembled reads =
Seed Input = batch
Reference sequence =
Variance detection =
Chloroplast sequence =
Dataset 1:
-----------------------
Read Length = 151
Insert size = 412
Platform = illumina
Single/Paired = PE
Combined reads =
Forward reads = batch
Reverse reads = batch
Heteroplasmy:
-----------------------
MAF =
HP exclude list =
PCR-free =
Optional:
-----------------------
Insert size auto = yes
Insert Range = 1.9
Insert Range strict = 1.3
Use Quality Scores = no
To run NOVOPlasty, make sure your batch, config, seed, and reads are in the same place, or specify their absolute paths.
###Simply type the line below. Note: you may need to type 'perl' before the script.
NOVOPlasty4.3.1.pl -c config.txt
2. Assemble, orient, and verify whole plastid genome sequences with Fast-Plast
3. Bait, map, and de novo assemble whole plastid genome sequences with GetOrganelle
Running GetOrganelle
General command format
get_organelle_from_reads.py -1 SRR5602600_1.fastq.gz -2 SRR5602600_2.fastq.gz -o SRR5602600-plastome -R 15 -F embplant_pt
A run with:
-s C_mac_mac_reference_F2095.fasta # seed file of a plastome in fasta format, from GenBank
-1 C_mac_mac_0161e_Ouray_CO/C_mac_mac_0161e_Ouray_CO_S22_L001_R1_001.fastq.gz # read 1
-2 C_mac_mac_0161e_Ouray_CO/C_mac_mac_0161e_Ouray_CO_S22_L001_R2_001.fastq.gz # read 2
-o get_organelle_output/C_mac_mac_0161e_Ouray_CO_t16_seeded # specify a new output directory
-t 20 # run on 20 cores, will speed up read mapping and (maybe) spades assembly with multiple kmer lengths
-R 25 # the number of extension iterations
-k 21,31,45,65,85 # different kmer sizes for assemblies with spades
-F embplant_pt # use the embryophyte plastome database
FULL COMMAND
- Also uses 'nohup' and '&' to make it run in the background
nohup get_organelle_from_reads.py -1 C_mac_mac_0161e_Ouray_CO/C_mac_mac_0161e_Ouray_CO_S22_L001_R1_001.fastq.gz -2 C_mac_mac_0161e_Ouray_CO/C_mac_mac_0161e_Ouray_CO_S22_L001_R2_001.fastq.gz -o get_organelle_output/C_mac_mac_0161e_Ouray_CO_t16_seeded -s C_mac_mac_reference_F2095.fasta -t 20 -R 25 -k 21,31,45,65,85 -F embplant_pt &
For a quick & dirty run (not recommended, only for exploratory purposes)
get_organelle_from_reads.py -1 C_mac_mac_0161e_Ouray_CO/C_mac_mac_0161e_Ouray_CO_S22_L001_R1_001.fastq.gz -2 C_mac_mac_0161e_Ouray_CO/C_mac_mac_0161e_Ouray_CO_S22_L001_R2_001.fastq.gz -o get_organelle_output/C_mac_mac_0161e_Ouray_CO_t16_seeded --fast -k 21, 55, 85, -F embplant_pt
GetOrganelle gives weird fasta headers, e.g.
>6736872_6685368_6749650-,6684792-,6738466_6736954_6730482_6741986_6733100_6749442_6738164_6728062_6687416_6732826_6739348_6666028_6...
ATTTATAGGATTCAAATAATCAAATAAAATAAAGATAGGCGGGTAATAACCTTATTTATGACAAGTTTCAAATTGGTAAAGTATACCCCTAGGATAAAGAAGAAGAAGGGGCTGAGAAAACTCGCAAGAAAA...
Here is a simple for-loop using awk to rename the fasta headers for multiple files with the file names:
for FILE in *.fasta;
do
awk '/^>/ {gsub(/.fa(sta)?$/,"",FILENAME);printf(">%s\n",FILENAME);next;} {print}' $FILE > changed_${FILE}
done
Output:
>CHN-Hubei-1980
ATTTATAGGATTCAAATAATCAAATAAAATAAAGATAGGCGGGTAATAACCTTATTTA...
How can I reorient the plastome output of GetOrganelle? Use ECuADOR.pl script
First, make a new directory called 'reorient_test_ecuador'
mkdir reorient_test_ecuador
### then, move your plastomes there with mv or cp command
### run the script:
perl ECuADOR.pl -i reorient_test_ecuador -w 1000 -f fasta -out test --ext fasta --save_regions ALL --orient TRUE --noIRs 2