Using a new database for ancient DNA

[danimelsz]

Hi

I am working with ancient DNA of frogs (Amphibia: Anura). I successfully assembled some mitochondrial genes using MITObim. However, I want to test the performance of GetOrganelle.

When I use the default database, I am unable to assemble any read using GetOrganelle. As such, I want to use a personal database, only including mitogenomes of phylogenetically close references of frogs.

$ get_organelle_from_reads.py -u '/home/danimelsz/Desktop/cymb_FINAL/0_assembly/3_fastqscreen/output2_tally_cymbalum.tagged_filter.fastq.gz' -F animal_mt -o 6_getorganelle --reduce-reads-for-coverage inf --max-reads inf -k 11,15 -w 30 -R 15

GetOrganelle v1.7.7.0

get_organelle_from_reads.py assembles organelle genomes from genome skimming data.
Find updates in https://github.com/Kinggerm/GetOrganelle and see README.md for more information.

Python 3.7.12 | packaged by conda-forge | (default, Oct 26 2021, 06:08:21)  [GCC 9.4.0]
PLATFORM: Linux linux 5.19.0-35-generic #36~22.04.1-Ubuntu SMP PREEMPT_DYNAMIC Fri Feb 17 15:17:25 UTC 2 x86_64 x86_64
PYTHON LIBS: GetOrganelleLib 1.7.7.0; numpy 1.21.6; sympy 1.10.1; scipy 1.7.3
DEPENDENCIES: Bowtie2 2.3.5.1; SPAdes 3.13.0; Blast 2.5.0
GETORG_PATH=/home/danimelsz/.GetOrganelle
SEED  DB: animal_mt 0.0.1
LABEL DB: animal_mt 0.0.1
WORKING DIR: /home/danimelsz/Desktop/cymb_FINAL/0_assembly/3_fastqscreen
/home/danimelsz/miniconda3/envs/getorganelle/bin/get_organelle_from_reads.py -u /home/danimelsz/Desktop/cymb_FINAL/0_assembly/3_fastqscreen/output2_tally_cymbalum.tagged_filter.fastq.gz -F animal_mt -o 6_getorganelle --reduce-reads-for-coverage inf --max-reads inf -k 11,15 -w 30 -R 15

2023-05-14 20:32:55,148 - INFO: Pre-reading fastq ...
2023-05-14 20:32:55,148 - INFO: Unzipping reads file: /home/danimelsz/Desktop/cymb_FINAL/0_assembly/3_fastqscreen/output2_tally_cymbalum.tagged_filter.fastq.gz (366419496 bytes)
2023-05-14 20:33:02,792 - INFO: Counting read qualities ...
2023-05-14 20:33:02,810 - INFO: Identified quality encoding format = Sanger
2023-05-14 20:33:02,810 - INFO: Phred offset = 33
2023-05-14 20:33:02,810 - INFO: Trimming bases with qualities (0.00%): 33..33  !
2023-05-14 20:33:02,814 - INFO: Mean error rate = 0.0003
2023-05-14 20:33:02,814 - INFO: Counting read lengths ...
2023-05-14 20:33:13,911 - INFO: Mean = 35.1 bp, maximum = 72 bp.
2023-05-14 20:33:13,911 - INFO: Reads used = 14480638
2023-05-14 20:33:13,911 - INFO: Pre-reading fastq finished.

2023-05-14 20:33:13,911 - INFO: Making seed reads ...
2023-05-14 20:33:13,912 - INFO: Seed bowtie2 index existed!
2023-05-14 20:33:13,912 - INFO: Mapping reads to seed bowtie2 index ...
2023-05-14 20:35:00,835 - INFO: Mapping finished.
2023-05-14 20:35:00,835 - INFO: Seed reads made: 6_getorganelle/seed/animal_mt.initial.fq (689590 bytes)
2023-05-14 20:35:00,836 - INFO: Making seed reads finished.

2023-05-14 20:35:00,836 - INFO: Checking seed reads and parameters ...
2023-05-14 20:35:02,405 - INFO: Estimated animal_mt-hitting base-coverage = 13.75
2023-05-14 20:35:02,567 - INFO: Setting '--max-extending-len inf'
2023-05-14 20:35:02,636 - INFO: Checking seed reads and parameters finished.

2023-05-14 20:35:02,637 - INFO: Making read index ...
2023-05-14 20:35:58,336 - INFO: 8894756 candidates in all 14480638 reads
2023-05-14 20:35:58,336 - INFO: Pre-grouping reads ...
2023-05-14 20:35:58,336 - INFO: Setting '--pre-w 30'
2023-05-14 20:35:58,944 - INFO: 61999/61999 used/duplicated
2023-05-14 20:36:02,393 - INFO: 2784 groups made.
2023-05-14 20:36:03,458 - INFO: Making read index finished.

2023-05-14 20:36:03,458 - INFO: Extending ...
2023-05-14 20:36:03,458 - INFO: Adding initial words ...
2023-05-14 20:36:03,476 - INFO: AW 14432
2023-05-14 20:36:20,898 - INFO: Round 1: 8894756/8894756 AI 754167 AW 3814518
2023-05-14 20:36:39,481 - INFO: Round 2: 8894756/8894756 AI 1901720 AW 8648337
2023-05-14 20:36:53,835 - INFO: Round 3: 8894756/8894756 AI 2231478 AW 10475109
2023-05-14 20:37:06,369 - INFO: Round 4: 8894756/8894756 AI 2352244 AW 11196649
2023-05-14 20:37:19,272 - INFO: Round 5: 8894756/8894756 AI 2406339 AW 11516741
2023-05-14 20:37:33,566 - INFO: Round 6: 8894756/8894756 AI 2432898 AW 11681463
2023-05-14 20:37:46,594 - INFO: Round 7: 8894756/8894756 AI 2451859 AW 11797639
2023-05-14 20:37:59,451 - INFO: Round 8: 8894756/8894756 AI 2464796 AW 11880645
2023-05-14 20:38:13,461 - INFO: Round 9: 8894756/8894756 AI 2474719 AW 11941015
2023-05-14 20:38:27,056 - INFO: Round 10: 8894756/8894756 AI 2480303 AW 11975085
2023-05-14 20:38:39,724 - INFO: Round 11: 8894756/8894756 AI 2483614 AW 11995579
2023-05-14 20:38:51,914 - INFO: Round 12: 8894756/8894756 AI 2485936 AW 12009195
2023-05-14 20:39:04,088 - INFO: Round 13: 8894756/8894756 AI 2487099 AW 12016159
2023-05-14 20:39:17,636 - INFO: Round 14: 8894756/8894756 AI 2488116 AW 12022459
2023-05-14 20:39:31,615 - INFO: Round 15: 8894756/8894756 AI 2489082 AW 12027665
2023-05-14 20:39:31,615 - INFO: Hit the round limit 15 and terminated ...
2023-05-14 20:39:49,495 - INFO: Extending finished.

2023-05-14 20:39:49,665 - INFO: Setting '-k '
2023-05-14 20:39:49,665 - INFO: Assembling using SPAdes ...
2023-05-14 20:39:49,671 - INFO: spades.py -t 1  --phred-offset 33 --s1 6_getorganelle/extended_1.fq  -o 6_getorganelle/extended_spades
2023-05-14 20:52:28,961 - INFO: Assembling finished.

2023-05-14 20:52:30,381 - WARNING: Slimming 6_getorganelle/extended_spades/K55/assembly_graph.fastg finished with no target organelle contigs found!
2023-05-14 20:52:30,382 - WARNING: No sequence hit our LabelDatabase!
2023-05-14 20:52:30,382 - WARNING: This might due to unreasonable seed/parameter choices or a bug.
2023-05-14 20:52:30,382 - INFO: Please open an issue at https://github.com/Kinggerm/GetOrganelle/issues with the get_org.log.txt file.


Total cost 1176.13 s
Thank you!

How can I edit the LabelDatabase and SeedDatabase that are located at ~./GetOrganelle/ ?

Could you provide me the commands for this task? I think Bowtie2 will be required to index them in the SeedDatabase. However, I do not know how to provide/edit the files in LabelDatabase, namely *.nhr *.nin *.nsq.

[JianjunJin]

You don't need to edit the files at ~./GetOrganelle. The short answer is that the key flags for using your own seed and label databases are -s and --genes, which both should be followed with a fasta file.

Please see here for step-by-step guidance to prepare these fasta files. You don't need to run bowtie2 or blast (for *.nhr etc) from your side.

Let me know if you have further questions.