novoalab / nano-trnaseq Goto Github PK

Hi developer!
I am so confused about the parameters in the part analysis_configuration.read_classification in the Minknow configure file (as showed in below picture), would you please tell the details what median, local_median, median_before,local_range ect. mean ? And how dose this setting work to classify read？

FYI for anyone else running on a Mac instead of Linux machine, the location for MinKNOW config files is different. The below should work for MacOS:

sudo rsync -a conf/package/flow_cells.toml /Applications/MinKNOW.app/Contents/Resources/conf/package
sudo rsync -a conf/package/sequencing/*.toml /Applications/MinKNOW.app/Contents/Resources/conf/package/sequencing

Thanks again for solving this short read RNA issue!

Hello,

This is a really lovely tool and an elegant solution to the problem of sequencing short reads by DRS. We have been testing this in our lab by performing 18-24 hrs sequencing runs, saving the bulk output, and then running the modified (short) analysis using the method you describe.

Initially, we would specify the run time for the simulation to be the same as the original run (e.g. 24 hrs) but we then started testing long run times in the simulation (e.g. 72 hrs) and observed a huge increase in the number of reads obtained (e.g. an increase from 500k at 24 hrs to 1500k at 72 hrs, despite the original run only being 18 hrs). Do you have any idea why this happens and did you observe the same for your own datasets?

Based on introduction, I updated the flow_cells.toml and copied the sequencing_MIN106_RNA_short.toml to /opt/ont/minknow/conf/package. However, my MinKNOW didn't recognize the sequencing_MIN106_RNA_short.toml with error message "Cannot be parsed correctly - MinKNOW version imcompatibility". My MinION is MC-114223 and MinKNOW version is 23.04.5 (bionic). There are a lot of difference between system sequencing_MIN106_RNA.toml and your sequencing_MIN106_RNA_short.toml. The system sequencing_MIN106_RNA.toml file attached. Would you please let me know how to modify the sequencing_MIN106_RNA_short.toml to make it compatible with my MinKNOW? Thank you so much.

minit@MC-114223:/opt/ont/minknow/conf/package/sequencing$ diff sequencing_MIN106_RNA.toml sequencing_MIN106_RNA_short.toml
10c10
< flow_cells = ["FLO-MIN106", "FLO-FLG001"]
---
> flow_cells = ["FLO-MIN106_short", "FLO-FLG001_short"]
19,22d18
< [meta.protocol.mk1c]
< default_basecall_model = "rna_r9.4.1_70bps_hac_mk1c.cfg"
< available_basecall_models = ["rna_r9.4.1_70bps_fast_mk1c.cfg", "rna_r9.4.1_70bps_hac_mk1c.cfg"]
<
28c24
< minknow_core = "5.5"
---
> minknow_core = "5.0"
51a48,50
> [basecaller_configuration.read_filtering]
> min_qscore = 7
>
88,89c87,88
<      "adapter=         (median_before,gt,160)&(median_before,lt,280)&(local_range,gt,5)&(local_range,lt,50)&(local_median,gt,50)&(local_median,lt,120)&(local_median_sd,gt,0.5)&(local_median_sd,lt,2.5)&(duration,lt,5)",
<      "strand=          (local_range,gt,25)&(local_range,lt,60)&(local_median,gt,60)&(local_median,lt,115)&(local_median_sd,gt,1)&(local_median_sd,lt,4)&(duration,gt,2)",
---
>      "adapter=         (median_before,gt,160)&(median_before,lt,280)&(local_range,gt,5)&(local_range,lt,50)&(local_median,gt,50)&(local_median,lt,120)&(local_median_sd,gt,0.5)&(local_median_sd,lt,2.5)&(duration,lt,2)",
>      "strand=          (local_range,gt,25)&(local_range,lt,60)&(local_median,gt,60)&(local_median,lt,115)&(local_median_sd,gt,1)&(local_median_sd,lt,4)&(duration,gt,1)",
125,126c124,126
< enable_relative_unblock_voltage = false
< effective_unblock_voltage = 110
---
> enable_relative_unblock_voltage = true
> unblock_voltage_gap = 480
> run_time = 172800 # (seconds) 1hr=3600
131a132,133
> #simulation="/path_to_bulk_dump.fast5"
>
183,184c185,186
< rest_duration  = [ 3, 3, 15, 30 ]
< repeats        = [ 1, 1, 4, 4 ]
---
> rest_duration  = [   3,   3,   15, 30   ]
> repeats        = [   1,   1,    4,  4   ]
224,227c226
< enabled = true
<
< interval = 7200 # Correct this often (seconds)
< reset_interval_every_mux_scan = true  # Only run every x seconds _inside_ a mux scan
---
> enabled = false
228a228
> interval = 5400 # Correct this often (seconds)
232,233d231
< minimum_voltage_adjustment = 5
<
270a269
> muxes = [1, 2, 3, 4]
296,297c295,296
< rest_duration  = [ 3, 3, 15, 30 ]
< repeats        = [ 1, 1, 4, 4 ]
---
> rest_duration  = [   3,   3,   15, 30   ]
> repeats        = [   1,   1,    4,  4   ]
328,329c327,328
< rest_duration  = [ 3, 3, 3, 3 ]
< repeats        = [ 1, 1, 4, 4 ]
---
> rest_duration  = [   3,   3,   3, 3   ]
> repeats        = [   1,   1,    4,  4   ]
348,364c347
< arguments = ["--guppy_config=rna_r9.4.1_70bps_fast.cfg", "--enrich_unblock_min_sequence_length=200", "--deplete_stop_receiving_min_sequence_length=4000"]
<
< [custom_processes.read_until.mk1c]
< arguments = ["--guppy_config=rna_r9.4.1_70bps_fast_mk1c.cfg", "--enrich_unblock_min_sequence_length=200", "--deplete_stop_receiving_min_sequence_length=4000"]
<
<
< ##################
< #   Run Until   #
< ##################
<
< [custom_processes.run_until]
< script = "sequencing/run_until/run_until_script.py"
< enabled = true
<
< # See the run until script argparse for parameters to pass
< # Anything passed by the UI will be on the end of this
< arguments = []
---
> arguments = ["--guppy_config=rna_r9.4.1_70bps_fast.cfg"]

sequencing_MIN106_RNA.toml.txt

We generated bulk fast5 files based on your introduction. Now we want to perform analysis in simulation mode. We installed the MinKnow standalone software downloaded from Nanopore website in another powerful workstation. When I started the MinKNOW UI, I found "My device" with "No positions connected" in connection manager. So I cannot do the analysis in simulation mode since there is no flowcell dropbox that I can select the alternative configuration. Do we have to import the alternative configuration files to the MinKNOW in the real MinION device? Thank you so much.

Hi @lpryszcz

congratulations on your manuscript !

I would like to go through your analysis that produces STable 4.
I have checked the EBI repository: https://www.ebi.ac.uk/ena/data/view/PRJEB55684
but there are no FASTQ files, only archives with FAST5 files.

Could you kindly share the FASTQ files for 3_NanotRNAseq_IVT+tRNAphe.tar.gz and 4_NanotRNAseq_IVT+tRNAphe.tar.gz with me ?

Thank you.

I'm trying to make changes to config file to permit short mode sequencing and/or simulation on a Promethion. A first naive attempt was to just try adding Promethion flow cells to flow_cells.toml and then add "FLO-PRO002_short" to sequencing_MIN106_RNA_short.toml. However, when we actually try to start a run with this short read mode, we get through the flow cell check and then almost immediately drop to zero pores. My suspicion is that one or more of the settings in this file (target temperature? min/max voltage?) are MinION specific and incompatible with Promethion settings, causing the pores to register as nonfunctional once the run starts.

Have you all played with settings for the Promethion at all? I shot an email to ONT customer support too, but that's always hit or miss.