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replikation / poreCov

SARS-CoV-2 workflow for nanopore sequence data
https://case-group.github.io/
GNU General Public License v3.0
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VarSkipV2b primer does not work as expected #246

Closed svonoy closed 1 year ago

svonoy commented 1 year ago

We switched from to VarSkip Short V2 Primer for the omicron variants. We tried to update our analyzing workflow. In poreCov we tried to simply replace "--primerV V3" to the new included primer from NEB, which should be available by: "--primerV VarSkipV2b".

Command used

Old command for delta variants (which worked):

nextflow run replikation/poreCov --fastq_pass /home/user/Schreibtisch/nextflow_Input/ \
  -r 0.9.5 --cores 31 --memory 32 -profile local,docker \
  --update --output /home/user/Schreibtisch/nextflow_Result/

Our new command whch results in error messages:

nextflow run replikation/poreCov --fastq_pass /home/user/Schreibtisch/nextflow_Input/ \
  -r 1.7.1 --primerV VarSkipV2b \
  --cores 31 --memory 32 -profile local,docker \
  --update --output /home/user/Schreibtisch/nextflow_Result/

Error code

Our new poreCov workflow produces this error message:

Could not generate any genomes, please check your reads /home/user/Schreibtisch/nextflow_Result//1.Read_quality
[33/52052f] NOTE: Process `artic_ncov_wf:artic_medaka (1)` terminated with an error exit status (20) -- Error is ignored

and most importantly we do not get our summary report

More Context

Computer specs if you are running locally (not in the cloud or a cluster)

OPERATING_SYSTEM = Ubuntu 20.4 Cores = 32 RAM = 64 GB

Additional Information

We are aware that our results may not be any good, but we just want to get the final summary report with the RKI reports. For tests we changed primerV to another primer, e.g. V4, the analysis was working "fine" and we got no error messages and the final summary report was generated (obviously the QC did not pass).

Additional error code

.command.log file

``` $ cat /work/nextflow-poreCov-user/33/520527*/.command.log [M::mm_idx_gen::0.005*1.51] collected minimizers [M::mm_idx_gen::0.008*5.29] sorted minimizers [M::main::0.008*5.28] loaded/built the index for 1 target sequence(s) [M::mm_mapopt_update::0.009*4.93] mid_occ = 3 [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 1 [M::mm_idx_stat::0.010*4.67] distinct minimizers: 5587 (99.93% are singletons); average occurrences: 1.004; average spacing: 5.332 [M::worker_pipeline::4.250*7.49] mapped 103098 sequences [M::main] Version: 2.17-r941 [M::main] CMD: minimap2 -a -x map-ont -t 31 external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12_filtered.fastq.gz [M::main] Real time: 4.253 sec; CPU: 31.849 sec; Peak RSS: 0.212 GB 2023-02-03 09:27:26.220905: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcudart.so.11.0'; dlerror: libcudart.so.11.0: cannot open shared object file: No such file or directory 2023-02-03 09:27:26.220962: I tensorflow/stream_executor/cuda/cudart_stub.cc:29] Ignore above cudart dlerror if you do not have a GPU set up on your machine. [09:27:27 - ValidArgs] Reads will be filtered to only those with RG tag: nCoV-2019_2 [09:27:27 - Predict] Reducing threads to 2, anymore is a waste. 2023-02-03 09:27:27.950631: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcuda.so.1'; dlerror: libcuda.so.1: cannot open shared object file: No such file or directory 2023-02-03 09:27:27.950692: W tensorflow/stream_executor/cuda/cuda_driver.cc:326] failed call to cuInit: UNKNOWN ERROR (303) 2023-02-03 09:27:27.950840: I tensorflow/stream_executor/cuda/cuda_diagnostics.cc:163] no NVIDIA GPU device is present: /dev/nvidia0 does not exist [09:27:27 - Predict] It looks like you are running medaka without a GPU and attempted to set a high number of threads. We have scaled this down to an optimal number. If you wish to improve performance please see https://nanoporetech.github.io/medaka/installation.html#improving-parallelism. [09:27:27 - Predict] Setting tensorflow inter/intra-op threads to 2/1. [09:27:27 - Predict] Processing region(s): MN908947.3:0-29903 [09:27:27 - Predict] Using model: /opt/conda/envs/artic/lib/python3.6/site-packages/medaka/data/r941_min_hac_g507_model.tar.gz. [09:27:27 - Predict] Processing 1 long region(s) with batching. 2023-02-03 09:27:28.006063: I tensorflow/core/platform/cpu_feature_guard.cc:142] This TensorFlow binary is optimized with oneAPI Deep Neural Network Library (oneDNN) to use the following CPU instructions in performance-critical operations: AVX2 AVX512F FMA To enable them in other operations, rebuild TensorFlow with the appropriate compiler flags. [09:27:28 - MdlStrTF] Model [09:27:28 - MdlStrTF] loading weights from /tmp/tmpauiixhs3/model/variables/variables [09:27:28 - Sampler] Initializing sampler for consensus of region MN908947.3:0-29903. [09:27:28 - PWorker] Running inference for 0.0M draft bases. [09:27:31 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 841-1423. [09:27:31 - Feature] Processed MN908947.3:841.0-1423.0 (median depth 1000.0) [09:27:31 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 1695-2249. [09:27:31 - Feature] Processed MN908947.3:1695.0-2249.0 (median depth 1000.0) [09:27:31 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 2483-3066. [09:27:31 - Feature] Processed MN908947.3:2483.0-3066.1 (median depth 662.0) [09:27:31 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 3326-3914. [09:27:32 - Feature] Processed MN908947.3:3326.0-3914.0 (median depth 576.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 4167-4742. [09:27:32 - Feature] Processed MN908947.3:4167.0-4742.0 (median depth 480.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 5006-5583. [09:27:32 - Feature] Processed MN908947.3:5006.0-5583.0 (median depth 1000.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 5794-6362. [09:27:32 - Feature] Processed MN908947.3:5794.0-6362.1 (median depth 1000.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 6561-7146. [09:27:32 - Feature] Processed MN908947.3:6561.0-7146.0 (median depth 771.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 7403-8001. [09:27:32 - Feature] Processed MN908947.3:7403.0-8001.0 (median depth 877.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 8245-8828. [09:27:32 - Feature] Processed MN908947.3:8245.0-8828.3 (median depth 438.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 9099-9666. [09:27:32 - Feature] Processed MN908947.3:9099.0-9666.0 (median depth 1000.0) [09:27:32 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 9842-10419. [09:27:33 - Feature] Processed MN908947.3:9842.0-10419.0 (median depth 1000.0) [09:27:33 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 10696-11267. [09:27:33 - Feature] Processed MN908947.3:10696.0-11267.0 (median depth 942.0) [09:27:33 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 11371-11942. [09:27:34 - Feature] Processed MN908947.3:11371.0-11942.0 (median depth 1000.0) [09:27:34 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 12201-12773. [09:27:34 - Feature] Processed MN908947.3:12201.0-12773.0 (median depth 1000.0) [09:27:34 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 13038-13620. [09:27:34 - Feature] Processed MN908947.3:13038.0-13620.1 (median depth 1000.0) [09:27:34 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 13874-14443. [09:27:35 - Feature] Processed MN908947.3:13874.0-14443.0 (median depth 1000.0) [09:27:35 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 14651-15216. [09:27:35 - Feature] Processed MN908947.3:14651.0-15216.0 (median depth 1000.0) [09:27:35 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 15442-16028. [09:27:35 - Feature] Processed MN908947.3:15442.0-16028.0 (median depth 1000.0) [09:27:35 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 16277-16864. [09:27:35 - Feature] Processed MN908947.3:16277.0-16864.0 (median depth 1000.0) [09:27:35 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 17060-17645. [09:27:35 - Feature] Processed MN908947.3:17060.0-17645.0 (median depth 2.0) [09:27:35 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 17889-18462. [09:27:35 - Feature] Processed MN908947.3:17889.0-18462.0 (median depth 877.0) [09:27:35 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 18712-19272. [09:27:36 - Feature] Processed MN908947.3:18712.0-19272.0 (median depth 628.0) [09:27:36 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 19488-20063. [09:27:36 - Feature] Processed MN908947.3:19488.0-20063.0 (median depth 20.0) [09:27:36 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 20340-20911. [09:27:37 - Feature] Processed MN908947.3:20340.0-20911.1 (median depth 1000.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 21192-21752. [09:27:37 - Feature] Processed MN908947.3:21192.0-21752.0 (median depth 854.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 21926-22509. [09:27:37 - Feature] Processed MN908947.3:21926.0-22509.0 (median depth 968.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 22749-23309. [09:27:37 - Feature] Processed MN908947.3:22749.0-23309.0 (median depth 9.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 23545-24128. [09:27:37 - Feature] Processed MN908947.3:23545.0-24128.0 (median depth 1000.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 24375-24947. [09:27:37 - Feature] Processed MN908947.3:24375.0-24947.0 (median depth 1000.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 25171-25753. [09:27:37 - Feature] Processed MN908947.3:25171.0-25753.0 (median depth 1000.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 25965-26524. [09:27:37 - Feature] Processed MN908947.3:25965.0-26524.0 (median depth 1000.0) [09:27:37 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 26682-27287. [09:27:38 - Feature] Processed MN908947.3:26682.0-27287.0 (median depth 1000.0) [09:27:38 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 27514-28075. [09:27:38 - Feature] Processed MN908947.3:27514.0-28075.0 (median depth 1000.0) [09:27:38 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 28219-28804. [09:27:38 - Feature] Processed MN908947.3:28219.0-28804.1 (median depth 1000.0) [09:27:38 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 29062-29643. [09:27:39 - Feature] Processed MN908947.3:29062.0-29643.0 (median depth 869.0) [09:27:39 - Sampler] Took 10.47s to make features. [09:27:39 - Sampler] Region MN908947.3:17060.0-17645.0 (593 positions) is smaller than inference chunk length 800, quarantining. [09:27:39 - Sampler] Region MN908947.3:19488.0-20063.0 (757 positions) is smaller than inference chunk length 800, quarantining. [09:27:39 - Sampler] Region MN908947.3:22749.0-23309.0 (638 positions) is smaller than inference chunk length 800, quarantining. 2023-02-03 09:27:40.265780: I tensorflow/compiler/mlir/mlir_graph_optimization_pass.cc:176] None of the MLIR Optimization Passes are enabled (registered 2) 2023-02-03 09:27:40.297114: I tensorflow/core/platform/profile_utils/cpu_utils.cc:114] CPU Frequency: 3099995000 Hz [09:27:41 - PWorker] Batches in cache: 2. [09:27:41 - PWorker] 33.3% Done (0.0/0.0 Mbases) in 12.7s [09:27:43 - PWorker] Processed 2 batches [09:27:43 - PWorker] All done, 3 remainder regions. [09:27:43 - Predict] Processing 3 short region(s). [09:27:43 - MdlStrTF] Model [09:27:43 - MdlStrTF] loading weights from /tmp/tmpauiixhs3/model/variables/variables [09:27:43 - Sampler] Initializing sampler for consensus of region MN908947.3:17060-17646. [09:27:43 - Sampler] Initializing sampler for consensus of region MN908947.3:19488-20064. [09:27:43 - PWorker] Running inference for 0.0M draft bases. [09:27:44 - Feature] Processed MN908947.3:17060.0-17645.0 (median depth 2.0) [09:27:44 - Feature] Processed MN908947.3:19488.0-20063.0 (median depth 20.0) [09:27:44 - Sampler] Took 0.80s to make features. [09:27:44 - Sampler] Took 0.88s to make features. [09:27:44 - Sampler] Initializing sampler for consensus of region MN908947.3:22749-23310. [09:27:44 - Feature] Processed MN908947.3:22749.0-23309.0 (median depth 9.0) [09:27:44 - Sampler] Took 0.23s to make features. [09:27:48 - PWorker] Batches in cache: 1. [09:27:48 - PWorker] Processed 3 batches [09:27:48 - PWorker] All done, 0 remainder regions. [09:27:48 - Predict] Finished processing all regions. 2023-02-03 09:27:49.713066: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcudart.so.11.0'; dlerror: libcudart.so.11.0: cannot open shared object file: No such file or directory 2023-02-03 09:27:49.713099: I tensorflow/stream_executor/cuda/cudart_stub.cc:29] Ignore above cudart dlerror if you do not have a GPU set up on your machine. [09:27:50 - DataIndx] Loaded 1/1 (100.00%) sample files. [09:27:50 - Variants] Processing MN908947.3:0-. [09:27:50 - TrimOlap] MN908947.3:1228.0-1423.0 and MN908947.3:1695.0-1902.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:2056.2-2249.0 and MN908947.3:2483.0-2696.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:2854.0-3066.1 and MN908947.3:3326.0-3559.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:3702.2-3914.0 and MN908947.3:4167.0-4399.48 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:4509.0-4742.0 and MN908947.3:5006.0-5204.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:5391.4-5583.0 and MN908947.3:5794.0-5995.2 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:6146.3-6362.1 and MN908947.3:6561.0-6771.2 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:6920.1-7146.0 and MN908947.3:7403.0-7599.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:7809.1-8001.0 and MN908947.3:8245.0-8479.2 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:8584.0-8828.3 and MN908947.3:9099.0-9301.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:9457.4-9666.0 and MN908947.3:9842.0-10049.3 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:10237.1-10419.0 and MN908947.3:10696.0-10906.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:11073.45-11267.0 and MN908947.3:11371.0-11577.6 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:11752.4-11942.0 and MN908947.3:12201.0-12401.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:12580.0-12773.0 and MN908947.3:13038.0-13239.4 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:13412.2-13620.1 and MN908947.3:13874.0-14069.2 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:14250.2-14443.0 and MN908947.3:14651.0-14850.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:15030.2-15216.0 and MN908947.3:15442.0-15622.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:15828.1-16028.0 and MN908947.3:16277.0-16471.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:16658.1-16864.0 and MN908947.3:17060.0-17645.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:17060.0-17645.0 and MN908947.3:17889.0-18088.0 cannot be concatenated as there is no overlap and they do not abut. /opt/conda/envs/artic/lib/python3.6/site-packages/medaka/labels.py:382: RuntimeWarning: divide by zero encountered in log10 q = -10 * np.log10(err) [09:27:50 - TrimOlap] MN908947.3:18250.4-18462.0 and MN908947.3:18712.0-18941.2 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:19034.2-19272.0 and MN908947.3:19488.0-20063.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:19488.0-20063.0 and MN908947.3:20340.0-20541.25 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:20722.23-20911.1 and MN908947.3:21192.0-21402.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:21552.14-21752.0 and MN908947.3:21926.0-22134.18 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:22301.2-22509.0 and MN908947.3:22749.0-23309.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:22749.0-23309.0 and MN908947.3:23545.0-23739.6 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:23928.5-24128.0 and MN908947.3:24375.0-24552.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:24756.2-24947.0 and MN908947.3:25171.0-25362.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:50 - TrimOlap] MN908947.3:25538.3-25753.0 and MN908947.3:25965.0-26158.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:51 - TrimOlap] MN908947.3:26318.3-26524.0 and MN908947.3:26682.0-26876.1 cannot be concatenated as there is no overlap and they do not abut. [09:27:51 - TrimOlap] MN908947.3:27082.1-27287.0 and MN908947.3:27514.0-27729.0 cannot be concatenated as there is no overlap and they do not abut. [09:27:51 - TrimOlap] MN908947.3:27888.4-28075.0 and MN908947.3:28219.0-28435.4 cannot be concatenated as there is no overlap and they do not abut. [09:27:51 - TrimOlap] MN908947.3:28596.4-28804.1 and MN908947.3:29062.0-29247.1 cannot be concatenated as there is no overlap and they do not abut. 2023-02-03 09:27:51.899614: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcudart.so.11.0'; dlerror: libcudart.so.11.0: cannot open shared object file: No such file or directory 2023-02-03 09:27:51.899651: I tensorflow/stream_executor/cuda/cudart_stub.cc:29] Ignore above cudart dlerror if you do not have a GPU set up on your machine. [09:27:52 - ValidArgs] Reads will be filtered to only those with RG tag: nCoV-2019_2 [09:27:52 - Annotate] Getting chrom coordinates [09:27:52 - Annotate] Processing chunk with coordinates: MN908947.3:1287-29510 2023-02-03 09:27:54.596892: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcudart.so.11.0'; dlerror: libcudart.so.11.0: cannot open shared object file: No such file or directory 2023-02-03 09:27:54.596922: I tensorflow/stream_executor/cuda/cudart_stub.cc:29] Ignore above cudart dlerror if you do not have a GPU set up on your machine. [09:27:55 - ValidArgs] Reads will be filtered to only those with RG tag: nCoV-2019_1 [09:27:55 - Predict] Reducing threads to 2, anymore is a waste. 2023-02-03 09:27:55.536004: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcuda.so.1'; dlerror: libcuda.so.1: cannot open shared object file: No such file or directory 2023-02-03 09:27:55.536032: W tensorflow/stream_executor/cuda/cuda_driver.cc:326] failed call to cuInit: UNKNOWN ERROR (303) 2023-02-03 09:27:55.536055: I tensorflow/stream_executor/cuda/cuda_diagnostics.cc:163] no NVIDIA GPU device is present: /dev/nvidia0 does not exist [09:27:55 - Predict] It looks like you are running medaka without a GPU and attempted to set a high number of threads. We have scaled this down to an optimal number. If you wish to improve performance please see https://nanoporetech.github.io/medaka/installation.html#improving-parallelism. [09:27:55 - Predict] Setting tensorflow inter/intra-op threads to 2/1. [09:27:55 - Predict] Processing region(s): MN908947.3:0-29903 [09:27:55 - Predict] Using model: /opt/conda/envs/artic/lib/python3.6/site-packages/medaka/data/r941_min_hac_g507_model.tar.gz. [09:27:55 - Predict] Processing 1 long region(s) with batching. 2023-02-03 09:27:55.567258: I tensorflow/core/platform/cpu_feature_guard.cc:142] This TensorFlow binary is optimized with oneAPI Deep Neural Network Library (oneDNN) to use the following CPU instructions in performance-critical operations: AVX2 AVX512F FMA To enable them in other operations, rebuild TensorFlow with the appropriate compiler flags. [09:27:56 - MdlStrTF] Model [09:27:56 - MdlStrTF] loading weights from /tmp/tmptiejfwd2/model/variables/variables [09:27:56 - Sampler] Initializing sampler for consensus of region MN908947.3:0-29903. [09:27:56 - PWorker] Running inference for 0.0M draft bases. [09:27:58 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 22-997. [09:27:58 - Feature] Processed MN908947.3:22.0-997.0 (median depth 999.0) [09:27:58 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 1277-1843. [09:27:58 - Feature] Processed MN908947.3:1277.0-1843.0 (median depth 116.0) [09:27:58 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 2061-2642. [09:27:59 - Feature] Processed MN908947.3:2061.0-2642.0 (median depth 1000.0) [09:27:59 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 2923-3491. [09:27:59 - Feature] Processed MN908947.3:2923.0-3491.0 (median depth 861.0) [09:27:59 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 3753-4321. [09:27:59 - Feature] Processed MN908947.3:3753.0-4321.0 (median depth 938.0) [09:27:59 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 4594-5167. [09:27:59 - Feature] Processed MN908947.3:4594.0-5167.0 (median depth 392.0) [09:27:59 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 5340-6010. [09:27:59 - Feature] Processed MN908947.3:5340.0-6010.0 (median depth 1000.0) [09:27:59 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 6217-6775. [09:27:59 - Feature] Processed MN908947.3:6217.0-6775.0 (median depth 378.0) [09:27:59 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 7000-7573. [09:28:00 - Feature] Processed MN908947.3:7000.0-7573.1 (median depth 1000.0) [09:28:00 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 7851-8369. [09:28:00 - Feature] Processed MN908947.3:7851.0-8369.0 (median depth 963.0) [09:28:00 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 8685-9242. [09:28:00 - Feature] Processed MN908947.3:8685.0-9242.0 (median depth 1000.0) [09:28:00 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 9478-10060. [09:28:01 - Feature] Processed MN908947.3:9478.0-10060.1 (median depth 790.0) [09:28:01 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 10269-10837. [09:28:01 - Feature] Processed MN908947.3:10269.0-10837.0 (median depth 981.0) [09:28:01 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 11092-11669. [09:28:01 - Feature] Processed MN908947.3:11092.0-11669.0 (median depth 1000.0) [09:28:01 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 11814-12352. [09:28:01 - Feature] Processed MN908947.3:11814.0-12352.0 (median depth 1000.0) [09:28:01 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 12632-13187. [09:28:02 - Feature] Processed MN908947.3:12632.0-13187.0 (median depth 1000.0) [09:28:02 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 13479-14030. [09:28:02 - Feature] Processed MN908947.3:13479.0-14030.1 (median depth 1000.0) [09:28:02 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 14290-14854. [09:28:02 - Feature] Processed MN908947.3:14290.0-14854.1 (median depth 1000.0) [09:28:02 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 15064-15621. [09:28:03 - Feature] Processed MN908947.3:15064.0-15621.0 (median depth 699.0) [09:28:03 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 15862-16442. [09:28:03 - Feature] Processed MN908947.3:15862.0-16442.0 (median depth 1000.0) [09:28:03 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 16689-17265. [09:28:03 - Feature] Processed MN908947.3:16689.0-17265.0 (median depth 832.0) [09:28:03 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 17476-18058. [09:28:03 - Feature] Processed MN908947.3:17476.0-18058.0 (median depth 8.0) [09:28:03 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 18311-18875. [09:28:03 - Feature] Processed MN908947.3:18311.0-18875.0 (median depth 1000.0) [09:28:03 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 19098-19654. [09:28:04 - Feature] Processed MN908947.3:19098.0-19654.0 (median depth 851.0) [09:28:04 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 19893-20483. [09:28:04 - Feature] Processed MN908947.3:19893.0-20483.2 (median depth 1000.0) [09:28:04 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 20766-21337. [09:28:04 - Feature] Processed MN908947.3:20766.0-21337.0 (median depth 934.0) [09:28:04 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 21537-22112. [09:28:05 - Feature] Processed MN908947.3:21537.0-22112.0 (median depth 1000.0) [09:28:05 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 22347-22876. [09:28:06 - Feature] Processed MN908947.3:22347.0-22876.0 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 23122-23707. [09:28:06 - Feature] Processed MN908947.3:23122.0-23707.0 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 23979-24540. [09:28:06 - Feature] Processed MN908947.3:23979.0-24540.0 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 24789-25352. [09:28:06 - Feature] Processed MN908947.3:24789.0-25352.0 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 25526-26142. [09:28:06 - Feature] Processed MN908947.3:25526.0-26142.0 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 26359-26926. [09:28:06 - Feature] Processed MN908947.3:26359.0-26926.1 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 27081-27670. [09:28:06 - Feature] Processed MN908947.3:27081.0-27670.0 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 27835-28399. [09:28:06 - Feature] Processed MN908947.3:27835.0-28399.0 (median depth 1000.0) [09:28:06 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 28627-29224. [09:28:07 - Feature] Processed MN908947.3:28627.0-29224.0 (median depth 1000.0) [09:28:07 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 29270-29830. [09:28:07 - Feature] Processed MN908947.3:29270.0-29830.0 (median depth 1000.0) [09:28:07 - Sampler] Took 11.67s to make features. [09:28:07 - Sampler] Region MN908947.3:17476.0-18058.0 (667 positions) is smaller than inference chunk length 800, quarantining. 2023-02-03 09:28:08.850885: I tensorflow/compiler/mlir/mlir_graph_optimization_pass.cc:176] None of the MLIR Optimization Passes are enabled (registered 2) 2023-02-03 09:28:08.876965: I tensorflow/core/platform/profile_utils/cpu_utils.cc:114] CPU Frequency: 3099995000 Hz [09:28:09 - PWorker] Batches in cache: 2. [09:28:09 - PWorker] 34.8% Done (0.0/0.0 Mbases) in 13.9s [09:28:11 - PWorker] Processed 2 batches [09:28:11 - PWorker] All done, 1 remainder regions. [09:28:11 - Predict] Processing 1 short region(s). [09:28:12 - MdlStrTF] Model [09:28:12 - MdlStrTF] loading weights from /tmp/tmptiejfwd2/model/variables/variables [09:28:12 - Sampler] Initializing sampler for consensus of region MN908947.3:17476-18059. [09:28:12 - PWorker] Running inference for 0.0M draft bases. [09:28:12 - Feature] Processed MN908947.3:17476.0-18058.0 (median depth 8.0) [09:28:12 - Sampler] Took 0.43s to make features. [09:28:14 - PWorker] Processed 1 batches [09:28:14 - PWorker] All done, 0 remainder regions. [09:28:14 - Predict] Finished processing all regions. 2023-02-03 09:28:15.121003: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcudart.so.11.0'; dlerror: libcudart.so.11.0: cannot open shared object file: No such file or directory 2023-02-03 09:28:15.121032: I tensorflow/stream_executor/cuda/cudart_stub.cc:29] Ignore above cudart dlerror if you do not have a GPU set up on your machine. [09:28:16 - DataIndx] Loaded 1/1 (100.00%) sample files. [09:28:16 - Variants] Processing MN908947.3:0-. [09:28:16 - TrimOlap] MN908947.3:803.0-997.0 and MN908947.3:1277.0-1635.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:1449.0-1843.0 and MN908947.3:2061.0-2254.3 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:2431.0-2642.0 and MN908947.3:2923.0-3129.3 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:3298.0-3491.0 and MN908947.3:3753.0-3948.1 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:4134.0-4321.0 and MN908947.3:4594.0-4833.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:4934.0-5167.0 and MN908947.3:5340.0-5544.1 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:5804.3-6010.0 and MN908947.3:6217.0-6490.2 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:6529.0-6775.0 and MN908947.3:7000.0-7181.2 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:7381.6-7573.1 and MN908947.3:7851.0-8047.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:8166.6-8369.0 and MN908947.3:8685.0-8873.4 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:9028.1-9242.0 and MN908947.3:9478.0-9692.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:9846.1-10060.1 and MN908947.3:10269.0-10461.0 cannot be concatenated as there is no overlap and they do not abut. /opt/conda/envs/artic/lib/python3.6/site-packages/medaka/labels.py:382: RuntimeWarning: divide by zero encountered in log10 q = -10 * np.log10(err) [09:28:16 - TrimOlap] MN908947.3:10622.3-10837.0 and MN908947.3:11092.0-11307.3 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:11481.3-11669.0 and MN908947.3:11814.0-12014.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:12160.3-12352.0 and MN908947.3:12632.0-12815.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:12992.1-13187.0 and MN908947.3:13479.0-13669.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:13824.4-14030.1 and MN908947.3:14290.0-14500.4 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:14667.1-14854.1 and MN908947.3:15064.0-15273.1 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:15402.2-15621.0 and MN908947.3:15862.0-16061.1 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:16239.3-16442.0 and MN908947.3:16689.0-16850.2 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:17058.4-17265.0 and MN908947.3:17476.0-18058.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:17476.0-18058.0 and MN908947.3:18311.0-18490.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:18680.2-18875.0 and MN908947.3:19098.0-19299.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:19449.1-19654.0 and MN908947.3:19893.0-20082.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:20275.0-20483.2 and MN908947.3:20766.0-20965.1 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:21129.4-21337.0 and MN908947.3:21537.0-21740.2 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:21890.1-22112.0 and MN908947.3:22347.0-22559.2 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:22679.1-22876.0 and MN908947.3:23122.0-23323.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:23515.3-23707.0 and MN908947.3:23979.0-24191.4 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:24325.0-24540.0 and MN908947.3:24789.0-24967.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:25153.1-25352.0 and MN908947.3:25526.0-25727.2 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:25938.3-26142.0 and MN908947.3:26359.0-26564.0 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:26719.3-26926.1 and MN908947.3:27081.0-27291.3 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:27499.2-27670.0 and MN908947.3:27835.0-28033.3 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:28171.3-28399.0 and MN908947.3:28627.0-28812.2 cannot be concatenated as there is no overlap and they do not abut. [09:28:16 - TrimOlap] MN908947.3:29023.2-29224.0 and MN908947.3:29270.0-29455.1 cannot be concatenated as there is no overlap and they do not abut. 2023-02-03 09:28:17.342400: W tensorflow/stream_executor/platform/default/dso_loader.cc:64] Could not load dynamic library 'libcudart.so.11.0'; dlerror: libcudart.so.11.0: cannot open shared object file: No such file or directory 2023-02-03 09:28:17.342431: I tensorflow/stream_executor/cuda/cudart_stub.cc:29] Ignore above cudart dlerror if you do not have a GPU set up on your machine. [09:28:18 - ValidArgs] Reads will be filtered to only those with RG tag: nCoV-2019_1 [09:28:18 - Annotate] Getting chrom coordinates [09:28:18 - Annotate] Processing chunk with coordinates: MN908947.3:240-29801 Suppress variant 22786 Suppress variant 22813 Lines total/split/realigned/skipped: 88/0/0/0 Note: the --sample option not given, applying all records regardless of the genotype The site MN908947.3:3037 overlaps with another variant, skipping... The site MN908947.3:7528 overlaps with another variant, skipping... The site MN908947.3:9534 overlaps with another variant, skipping... The site MN908947.3:10029 overlaps with another variant, skipping... The site MN908947.3:14408 overlaps with another variant, skipping... The site MN908947.3:19955 overlaps with another variant, skipping... The site MN908947.3:21618 overlaps with another variant, skipping... The site MN908947.3:21632 overlaps with another variant, skipping... The site MN908947.3:21987 overlaps with another variant, skipping... The site MN908947.3:23599 overlaps with another variant, skipping... The site MN908947.3:23604 overlaps with another variant, skipping... The site MN908947.3:24424 overlaps with another variant, skipping... The site MN908947.3:24469 overlaps with another variant, skipping... The site MN908947.3:25584 overlaps with another variant, skipping... The site MN908947.3:26060 overlaps with another variant, skipping... The site MN908947.3:26709 overlaps with another variant, skipping... The site MN908947.3:27889 overlaps with another variant, skipping... The site MN908947.3:28271 overlaps with another variant, skipping... The site MN908947.3:28311 overlaps with another variant, skipping... The site MN908947.3:28330 overlaps with another variant, skipping... The site MN908947.3:28361 overlaps with another variant, skipping... The site MN908947.3:28777 overlaps with another variant, skipping... The site MN908947.3:29510 overlaps with another variant, skipping... Applied 65 variants MUSCLE v3.8.1551 by Robert C. Edgar http://www.drive5.com/muscle This software is donated to the public domain. Please cite: Edgar, R.C. Nucleic Acids Res 32(5), 1792-97. barcode12.muscle.in 2 seqs, lengths min 29844, max 29903, avg 29873 00:00:00 16 MB(1%) Iter 1 33.33% K-mer dist pass 1 00:00:00 16 MB(1%) Iter 1 100.00% K-mer dist pass 1 00:00:00 16 MB(1%) Iter 1 33.33% K-mer dist pass 2 00:00:00 16 MB(1%) Iter 1 100.00% K-mer dist pass 2 00:00:00 33 MB(3%) Iter 1 100.00% Align node 00:00:13 998 MB(91%) Iter 1 100.00% Align node 00:00:13 998 MB(91%) Iter 1 50.00% Root alignment 00:00:13 998 MB(91%) Iter 1 100.00% Root alignment 00:00:13 998 MB(91%) Iter 1 100.00% Root alignment in correct numbers of primer for varskip-0317-1_49 Running: minimap2 -a -x map-ont -t 31 external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12_filtered.fastq.gz | samtools view -bS -F 4 - | samtools sort -o barcode12.sorted.bam - Running: samtools index barcode12.sorted.bam Running: align_trim --normalise 500 external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.scheme.bed --start --remove-incorrect-pairs --report barcode12.alignreport.txt < barcode12.sorted.bam 2> barcode12.alignreport.er | samtools sort -T barcode12 - -o barcode12.trimmed.rg.sorted.bam Running: align_trim --normalise 500 external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.scheme.bed --remove-incorrect-pairs --report barcode12.alignreport.txt < barcode12.sorted.bam 2> barcode12.alignreport.er | samtools sort -T barcode12 - -o barcode12.primertrimmed.rg.sorted.bam Running: samtools index barcode12.trimmed.rg.sorted.bam Running: samtools index barcode12.primertrimmed.rg.sorted.bam Running: medaka consensus --model r941_min_hac_g507 --threads 31 --chunk_len 800 --chunk_ovlp 400 --RG nCoV-2019_2 barcode12.trimmed.rg.sorted.bam barcode12.nCoV-2019_2.hdf Running: medaka variant external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12.nCoV-2019_2.hdf barcode12.nCoV-2019_2.vcf Running: medaka tools annotate --pad 25 --RG nCoV-2019_2 barcode12.nCoV-2019_2.vcf external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12.trimmed.rg.sorted.bam barcode12.medaka-annotate.vcf Running: mv barcode12.medaka-annotate.vcf barcode12.nCoV-2019_2.vcf Running: medaka consensus --model r941_min_hac_g507 --threads 31 --chunk_len 800 --chunk_ovlp 400 --RG nCoV-2019_1 barcode12.trimmed.rg.sorted.bam barcode12.nCoV-2019_1.hdf Running: medaka variant external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12.nCoV-2019_1.hdf barcode12.nCoV-2019_1.vcf Running: medaka tools annotate --pad 25 --RG nCoV-2019_1 barcode12.nCoV-2019_1.vcf external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12.trimmed.rg.sorted.bam barcode12.medaka-annotate.vcf Running: mv barcode12.medaka-annotate.vcf barcode12.nCoV-2019_1.vcf Running: artic_vcf_merge barcode12 external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.scheme.bed 2> barcode12.primersitereport.txt nCoV-2019_2:barcode12.nCoV-2019_2.vcf nCoV-2019_1:barcode12.nCoV-2019_1.vcf Running: bgzip -f barcode12.merged.vcf Running: tabix -p vcf barcode12.merged.vcf.gz Running: artic-tools check_vcf --summaryOut barcode12.vcfreport.txt barcode12.merged.vcf.gz external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.scheme.bed 2> barcode12.vcfcheck.log Running: artic_vcf_filter --medaka --min-depth 20 --min-qual 20 --nanopolish-qual-cov-ratio 3.0 barcode12.merged.vcf.gz barcode12.pass.vcf barcode12.fail.vcf Running: artic_make_depth_mask --depth 20 external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12.primertrimmed.rg.sorted.bam barcode12.coverage_mask.txt Running: artic_mask external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta barcode12.coverage_mask.txt barcode12.fail.vcf barcode12.preconsensus.fasta Running: bcftools norm --check-ref x --fasta-ref barcode12.preconsensus.fasta -O z -o barcode12.pass.vcf.gz barcode12.pass.vcf Running: tabix -p vcf barcode12.pass.vcf.gz Running: bcftools consensus -f barcode12.preconsensus.fasta barcode12.pass.vcf.gz -m barcode12.coverage_mask.txt -o barcode12.consensus.fasta Running: artic_fasta_header barcode12.consensus.fasta "barcode12/ARTIC/medaka" Running: cat barcode12.consensus.fasta external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.reference.fasta > barcode12.muscle.in.fasta Running: muscle -in barcode12.muscle.in.fasta -out barcode12.muscle.out.fasta Running: artic_get_stats --min-depth 20 --scheme external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.scheme.bed --align-report barcode12.alignreport.txt --vcf-report barcode12.vcfreport.txt barcode12 Command failed:artic_get_stats --min-depth 20 --scheme external_primer_schemes/nCoV-2019/VarSkipV2b/nCoV-2019.scheme.bed --align-report barcode12.alignreport.txt --vcf-report barcode12.vcfreport.txt barcode12 ```

replikation commented 1 year ago

hi, thanks for reporting.

the varskip2b primers we provide are these, are these the correct ones?

as an alternative, you could try providing the .bed file for the primers by yourself (--primerV primers.bed, Formating info here) if you suspect some issues on this end. otherwise, it's difficult for me to figure out what happened without your fastq files.

Is poreCov producing .bam files in your output-dir? you can take a look at these with e.g. ugene to visually inspect how and if reads are mapped against the sarscov2 genome.

svonoy commented 1 year ago

Hi, thanks for your quick answer. We talked with NEB about our primers and we figured out that we actually used the VarSkipV2a primers instead of VarSkipV2b. We just ran the analysis again with the VarSkipV2 primer provided within this repository and suddenly the workflow ran perfectly. Our barcode even passed quality control. Regarding the .bam files, we dont have the ugene tool to inspect these files. In our faulty runs with the VarSkipV2b primer .bam files were generated, but compared to the new VarSkipV2(a) run they were missing .bam files of the name format "barcode_mapped_MN90.primertrimmed.sorted.bam". Is this the reason the workflow could not advance and we did not get our final summary report? This was our primary concern.

replikation commented 1 year ago

alright perfect

svonoy commented 1 year ago

At the end of last week we did a new sequencing run with VarSkip Short V2 primer + spike ins => VarSkipV2b. Sadly the poreCov run ended with the exact same error as described above. Analyzing with the VarSkipV2 primer still works fine. We also tried the poreCov workflow with custom primer files and used the the primer files from NEB directly. This too did not change the outcome of the analyzing run.

replikation commented 1 year ago

could you provide me your data via, e.g. mail otherwise, it's difficult for me to identify whats going wrong.

DataSpott commented 1 year ago

@svonoy Corrected the primer scheme. The issue was that the alternative primers need to be added as extra primer-pair. ARTIC recognizes only pairs of 2 primers. So instead of adding for example "_varskip-0317-1_49_RIGHTALT1" as a third primer to pair 49, you add it as "_varskip-0317-1_75RIGHT" (here just added it as the last pair to the scheme). And add the according "_varskip-0317-1_49LEFT" as "_varskip-0317-1_75LEFT".

svonoy commented 1 year ago

Works perfectly now! Thanks for the quick solution.