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Example 2
Finished the first example, let's try a little different run with a user-defined seed. We use the same dataset to assemble the Arabidopsis thaliana chloroplast genome.
Let's download the reads using wget (skip this step if you already have it for Example 1):
wget https://github.com/Kinggerm/GetOrganelleGallery/raw/master/Test/reads/Arabidopsis_simulated.1.fq.gz
wget https://github.com/Kinggerm/GetOrganelleGallery/raw/master/Test/reads/Arabidopsis_simulated.2.fq.gz
# use gitlab links if above links fail
# wget https://gitlab.com/Kinggerm/GetOrganelleGallery/-/raw/master/Test/reads/Arabidopsis_simulated.1.fq.gz
# wget https://gitlab.com/Kinggerm/GetOrganelleGallery/-/raw/master/Test/reads/Arabidopsis_simulated.2.fq.gz
check the integrity of downloaded file using md5sum:
md5sum Arabidopsis_simulated.*.fq.gz
# 935589bc609397f1bfc9c40f571f0f19 Arabidopsis_simulated.1.fq.gz
# d0f62eed78d2d2c6bed5f5aeaf4a2c11 Arabidopsis_simulated.2.fq.gz
# Please re-download the reads if md5 values unmatched
download the seed - the complete plastome of the common bean (Phaseolus vulgaris L.):
wget https://github.com/Kinggerm/GetOrganelleGallery/raw/master/Test/seed/Phaseolus_vulgaris_plastome.fasta
then do the plastome assembly:
get_organelle_from_reads.py -1 Arabidopsis_simulated.1.fq.gz -2 Arabidopsis_simulated.2.fq.gz -t 1 -o Arabidopsis_simulated.plastome -F embplant_pt -R 10 -s Phaseolus_vulgaris_plastome.fasta
# Flag Value Illustration
# -1 Arabidopsis_simulated.1.fq.gz Input file with the forward paired-end reads (*.fq/.gz/.tar.gz)
# -2 Arabidopsis_simulated.2.fq.gz Input file with the reverse paired-end reads (*.fq/.gz/.tar.gz)
# -t 1 Maximum threads to use. Default: 1
# -o Arabidopsis_simulated.plastome Output directory
# -F embplant_pt Target organelle genome type(s)
# -R 10 Maximum extension rounds
# -s Phaseolus_vulgaris_plastome.fasta User defined initial seed file
GetOrganelle v1.7.0-beta3 # file names changed according to 1.7.4-pre
get_organelle_from_reads.py assembles organelle genomes from genome skimming data.
Find updates inhttps://github.com/Kinggerm/GetOrganelleand see README.md for more information.Python 3.6.10 |Anaconda, Inc.| (default, Mar 25 2020, 23:51:54) [GCC 7.3.0]
PYTHON LIBS: numpy 1.18.1; sympy 1.5.1; scipy 1.4.1; psutil 5.7.0
DEPENDENCIES: Bowtie2 2.3.5.1; SPAdes 3.12.0; Blast 2.9.0; Bandage 0.8.1
WORKING DIR: /home/data1
/root/.pyenv/versions/miniconda3-4.3.30/bin/get_organelle_from_reads.py -1 Arabidopsis_simulated.1.fq.gz -2 Arabidopsis_simulated.2.fq.gz -t 1 -o Arabidopsis_simulated.plastome-user.seed -F embplant_pt -R 10 -s Phaseolus_vulgaris_plastome.fasta2020-05-25 07:18:24,365 - INFO: Pre-reading fastq ...
2020-05-25 07:18:24,365 - INFO: Estimating reads to use ... (to use all reads, set '--reduce-reads-for-coverage inf')
2020-05-25 07:18:25,126 - INFO: Estimating reads to use finished.
2020-05-25 07:18:25,126 - INFO: Unzipping reads file: Arabidopsis_simulated.1.fq.gz (8796915 bytes)
2020-05-25 07:18:25,299 - INFO: Unzipping reads file: Arabidopsis_simulated.2.fq.gz (9067061 bytes)
2020-05-25 07:18:25,469 - INFO: Counting read qualities ...
2020-05-25 07:18:25,594 - INFO: Identified quality encoding format = Illumina 1.8+
2020-05-25 07:18:25,595 - INFO: Trimming bases with qualities (0.00%): 33..33 !
2020-05-25 07:18:25,641 - INFO: Mean error rate = 0.0019
2020-05-25 07:18:25,642 - INFO: Counting read lengths ...
2020-05-25 07:18:25,850 - INFO: Mean = 150.0 bp, maximum = 150 bp.
2020-05-25 07:18:25,851 - INFO: Reads used = 91563+91563
2020-05-25 07:18:25,851 - INFO: Pre-reading fastq finished.2020-05-25 07:18:25,851 - INFO: Making seed reads ...
2020-05-25 07:18:25,858 - INFO: Making seed - bowtie2 index ...
2020-05-25 07:18:26,087 - INFO: Making seed - bowtie2 index finished.
2020-05-25 07:18:26,088 - INFO: Mapping reads to seed bowtie2 index ...
2020-05-25 07:18:29,328 - INFO: Mapping finished.
2020-05-25 07:18:29,329 - INFO: Seed reads made: Arabidopsis_simulated.plastome-user.seed/seed/embplant_pt.initial.fq (3794476 bytes)
2020-05-25 07:18:29,329 - INFO: Making seed reads finished.2020-05-25 07:18:29,331 - INFO: Checking seed reads and parameters ...
2020-05-25 07:18:29,331 - INFO: The automatically-estimated parameter(s) do not ensure the best choice(s).
2020-05-25 07:18:29,332 - INFO: If the result graph is not a circular organelle genome,
2020-05-25 07:18:29,332 - INFO: you could adjust the value(s) of '-w'/'-R' for another new run.
2020-05-25 07:18:30,558 - INFO: Pre-assembling mapped reads ...
2020-05-25 07:18:33,273 - INFO: Pre-assembling mapped reads finished.
2020-05-25 07:18:33,273 - INFO: Estimated embplant_pt-hitting base-coverage = 34.26
2020-05-25 07:18:33,273 - INFO: Estimated word size(s): 79
2020-05-25 07:18:33,273 - INFO: Setting '-w 79'
2020-05-25 07:18:33,274 - INFO: Setting '--max-extending-len inf'
2020-05-25 07:18:33,288 - INFO: Checking seed reads and parameters finished.2020-05-25 07:18:33,288 - INFO: Making read index ...
2020-05-25 07:18:34,440 - INFO: Mem 0.181 G, 178623 candidates in all 183126 reads
2020-05-25 07:18:34,441 - INFO: Pre-grouping reads ...
2020-05-25 07:18:34,441 - INFO: Setting '--pre-w 79'
2020-05-25 07:18:34,455 - INFO: Mem 0.171 G, 4074/4074 used/duplicated
2020-05-25 07:18:34,808 - INFO: Mem 0.192 G, 384 groups made.
2020-05-25 07:18:34,817 - INFO: Making read index finished.2020-05-25 07:18:34,817 - INFO: Extending ...
2020-05-25 07:18:34,817 - INFO: Adding initial words ...
2020-05-25 07:18:35,213 - INFO: AW 325138
2020-05-25 07:18:36,979 - INFO: Round 1: 178623/178623 AI 27052 AW 617812 Mem 0.228
2020-05-25 07:18:38,286 - INFO: Round 2: 178623/178623 AI 34363 AW 727022 Mem 0.257
2020-05-25 07:18:39,422 - INFO: Round 3: 178623/178623 AI 36904 AW 764534 Mem 0.262
2020-05-25 07:18:40,548 - INFO: Round 4: 178623/178623 AI 38202 AW 786764 Mem 0.265
2020-05-25 07:18:41,708 - INFO: Round 5: 178623/178623 AI 38931 AW 798264 Mem 0.266
2020-05-25 07:18:42,776 - INFO: Round 6: 178623/178623 AI 39209 AW 803430 Mem 0.267
2020-05-25 07:18:43,881 - INFO: Round 7: 178623/178623 AI 39488 AW 808318 Mem 0.268
2020-05-25 07:18:44,968 - INFO: Round 8: 178623/178623 AI 39548 AW 808688 Mem 0.268
2020-05-25 07:18:46,055 - INFO: Round 9: 178623/178623 AI 39548 AW 808688 Mem 0.268
2020-05-25 07:18:46,056 - INFO: No more reads found and terminated ...
2020-05-25 07:18:46,433 - INFO: Extending finished.2020-05-25 07:18:46,439 - INFO: Separating extended fastq file ...
2020-05-25 07:18:46,557 - INFO: Setting '-k 21,55,85,115'
2020-05-25 07:18:46,558 - INFO: Assembling using SPAdes ...
2020-05-25 07:18:46,564 - INFO: spades.py -t 1 -1 Arabidopsis_simulated.plastome-user.seed/extended_1_paired.fq -2 Arabidopsis_simulated.plastome-user.seed/extended_2_paired.fq --s1 Arabidopsis_simulated.plastome-user.seed/extended_1_unpaired.fq --s2 Arabidopsis_simulated.plastome-user.seed/extended_2_unpaired.fq -k 21,55,85,115 -o Arabidopsis_simulated.plastome-user.seed/extended_spades
2020-05-25 07:19:10,083 - INFO: Insert size = 499.35, deviation = 9.89013, left quantile = 487, right quantile = 512
2020-05-25 07:19:10,083 - INFO: Assembling finished.2020-05-25 07:19:11,193 - INFO: Slimming Arabidopsis_simulated.plastome-user.seed/extended_spades/K115/assembly_graph.fastg finished!
2020-05-25 07:19:12,236 - INFO: Slimming Arabidopsis_simulated.plastome-user.seed/extended_spades/K85/assembly_graph.fastg finished!
2020-05-25 07:19:13,262 - INFO: Slimming Arabidopsis_simulated.plastome-user.seed/extended_spades/K55/assembly_graph.fastg finished!
2020-05-25 07:19:13,263 - INFO: Slimming assembly graphs finished.2020-05-25 07:19:13,263 - INFO: Extracting embplant_pt from the assemblies ...
2020-05-25 07:19:13,264 - INFO: Disentangling Arabidopsis_simulated.plastome-user.seed/extended_spades/K115/assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg as a circular genome ...
2020-05-25 07:19:13,508 - INFO: Vertex_2910 #copy = 1
2020-05-25 07:19:13,508 - INFO: Vertex_2936 #copy = 2
2020-05-25 07:19:13,508 - INFO: Vertex_2938 #copy = 1
2020-05-25 07:19:13,509 - INFO: Average embplant_pt kmer-coverage = 10.0
2020-05-25 07:19:13,509 - INFO: Average embplant_pt base-coverage = 41.5
2020-05-25 07:19:13,509 - INFO: Writing output ...
2020-05-25 07:19:13,558 - WARNING: More than one circular genome structure produced ...
2020-05-25 07:19:13,558 - WARNING: Please check the final result to confirm whether they are simply flip-flop configurations!
2020-05-25 07:19:14,308 - INFO: Detecting large repeats (>1000 bp) in PATH1 with IRs detected, Total:LSC:SSC:Repeat(bp) = 154478:84170:17780:26264
2020-05-25 07:19:14,308 - INFO: Writing PATH1 of complete embplant_pt to Arabidopsis_simulated.plastome-user.seed/embplant_pt.K115.complete.graph1.1.path_sequence.fasta
2020-05-25 07:19:14,311 - INFO: Writing PATH2 of complete embplant_pt to Arabidopsis_simulated.plastome-user.seed/embplant_pt.K115.complete.graph1.2.path_sequence.fasta
2020-05-25 07:19:14,311 - INFO: Writing GRAPH to Arabidopsis_simulated.plastome-user.seed/embplant_pt.K115.complete.graph1.selected_graph.gfa
2020-05-25 07:19:14,485 - INFO: Writing GRAPH image to Arabidopsis_simulated.plastome-user.seed/embplant_pt.K115.complete.graph1.selected_graph.png
2020-05-25 07:19:14,485 - INFO: Result status of embplant_pt: circular genome
2020-05-25 07:19:14,501 - INFO: Please check the produced assembly image or manually visualize Arabidopsis_simulated.plastome-user.seed/extended_K115.assembly_graph.fastg.extend-embplant_pt-embplant_mt.fastg using Bandage to confirm the final result.
2020-05-25 07:19:14,502 - INFO: Writing output finished.
2020-05-25 07:19:14,502 - INFO: Extracting embplant_pt from the assemblies finished.
Total cost 50.65 s
Thank you!
Example 2 generated the sample files/folders in the first level directory as in Example 1, see Example 1 - Output files for a detailed illustration of output files.
Let's compare the result of these two runs:
diff Arabidopsis_simulated.plastome/embplant_pt.K115.complete.graph1.1.path_sequence.fasta Arabidopsis_simulated.plastome-user.seed/embplant_pt.K115.complete.graph1.1.path_sequence.fasta
1c1
< >3160-,3158-,3128-,3158+(circular)
---
> >2938-,2936-,2910-,2936+(circular)
They generated exactly the same sequence (only with different head names).