13
votes
Accepted
How can I improve the yield of MinION sequencing runs?
I attended a talk by Josh Quick at PoreCampAU 2017, in which he discussed some common barriers to getting both good sequencing yield and long read length. It mostly boils down to being more careful ...
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8
votes
Accepted
Is it possible to perform MinION sequencing offline?
Short answer: yes, but you need to get permission (and modified software) from ONT before doing that.
... but that doesn't tell the whole story. This question has the potential to be very confusing, ...
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8
votes
Accepted
Total reads aligning to each reference within a bam file
The quick way to get the number of alignments on each reference is
samtools idxstats my_bam.bam
Number of reads on each reference is column 3. Although, as has ...
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7
votes
What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?
There are also third party free and open source basecallers that haven't been developed by Oxford Nanopore.
Of particular note is Chiron, which gave the best uncorrected assembly identity among the ...
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6
votes
Accepted
Can I stop my nanopore sequencing run if there are no more reads being produced?
Yes, you can click "stop acquisition" and your run won't be negatively affected. All of the reads are saved as they are generated. I am not sure how this will impact live basecalling though if that is ...
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6
votes
Accepted
What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?
First of all - yes, you can generate FAST5 files and basecall later. Basecalling during the sequencing run is useful if you want results more quickly. You can also recall your FAST5 files with ...
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6
votes
Accepted
How does MinKNOW classify 1D reads as "pass" or "fail"?
The classification is indeed based purely on comparing a mean to a threshold... but it's not the mean of the Phred scores that represent base quality on a logarithmic scale, but rather the mean of the ...
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5
votes
Is it possible to perform MinION sequencing offline?
In short, yes offline basecalling is supported.
Oxford nanopore has made available an official offline basecaller, called albacore. If you have an account to the nanpore community website you can ...
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5
votes
What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?
It's worth noting two things as of Dec 2018:
Albacore is being deprecated (but is still available from the Nanopore developer portal).
Guppy is under active development, so Ryan Wick's comparisons ...
5
votes
Accepted
Demultiplex nanopore reads with custom barcodes
I've written some Perl code for demultiplexing nanopore reads using LAST. A workflow demonstrating the use of this script (and how it works under the hood) can be found here:
https://dx.doi.org/10....
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4
votes
Accepted
Can we use non base-called fast5 files in poretools?
No, poretools does not do basecalling. The poretools fastq command can be used to extract the FASTQ information from the basecalled FAST5 file (via MinKNOW live-...
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4
votes
Accepted
Can I use my computer while MinION is sequencing without negatively affecting the run?
Yes, that should be fine, especially if basecalling is disabled. Locking a screen should be fine as well.
I prefer to do basecalling after a run anyway, because there's a chance that it could chew up ...
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4
votes
Accepted
Nanopore Flongle vs MinION
For us, R10.4.1 Flongle flow cells are running at reduced yield due to lower numbers of working pores (something like 10-20 pores instead of 30-60 pores for R9.4.1 flow cells). This doesn't really ...
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3
votes
Extracting strand specific reads from MinION cDNA-PCR protocol
This has been set up as a bioinformatics protocol on protocols.io.
I've been using LAST to identify the adapter orientation relative to the genome, and then using that information to split BAM files ...
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3
votes
Accepted
How many reads has my sequencing run produced on minion?
That's indeed the number of reads and that's quite low. How was your pore occupancy (number of pores sequencing) and your flow cell QC (number of pores good enough for sequencing)? How was your ...
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3
votes
Accepted
Set directory where MinKNOW writes FAST5 files
I found a page explaining where this is set up in a user_conf file here:
https://community.nanoporetech.com/support/faq/test1/minknow/minknow/how-do-i-change-the-...
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3
votes
Accepted
Minion channel ID's from Albacore
There are actually 2048 usable sequencing wells, hexagonally packed with four wells connected to the same sequencing sensor/channel via a multiplex (mux) selector. The combination of the mux and the ...
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3
votes
Accepted
How do I find the most similar sequences from a large set of short sequences?
130k * 20bp is a small data set. At this scale, SSE2 Smith-Waterman may work well:
...
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3
votes
Demultiplex nanopore reads with custom barcodes
There are two other ways in which you could demultiplex reads if guppy doesn't have the config file for the kit you are using.
Minibar: This works for dual index barcodes specifically
Porechop: This ...
3
votes
Accepted
Microbial Classification with 16S Nanopore Data
Use kraken2 with one of the 16S databases. Kraken2 has worked well for me with nanopore sequences. Pavian can be used to inspect the Kraken2 report files visually.
Using uncorrected called reads has ...
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2
votes
How do I find the most similar sequences from a large set of short sequences?
For this application, you could probably also do something like calculate the Hamming distances between all of the strings in an all-vs-all approach (it should not take too long or too much overhead). ...
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2
votes
Accepted
Does MinKNOW work with Mac OSX high sierra 10.13.1?
Some replies from ONT is:
OS X is supported and you can view the installation instructions on
our Downloads page linked below. At the moment High Sierra is not
officially supported as we have ...
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2
votes
Accepted
ENA minion 2D data doesn't actually have 2D data
Probably not. ENA doesn't have any ability to specifically flag a file as 2D, and the nanopore file formats have changed a lot, so you pretty much need to rely on the submitter to separate out the ...
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2
votes
Accepted
What to do with the configuration test cell after configuring the MinION with it?
I would keep it, in case you need to do another configuration later on. I have used mine for that purpose.
I think storage doesn't really matter, I just put it in a drawer somewhere. Keep it away ...
- 1,314
2
votes
Albacore wheel not supported on this platform (Mac OS X El Capitan)
Not sure about conda as I don't use it, but I can highly recommend pipenv which is the python.org recommended package management ...
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2
votes
How can I improve the yield of MinION sequencing runs?
My bet is that the nanopore folks have (of course!) done a ton of optimisation on two key things:
The DNA cleanup
The library preps
I think that for most DNA extractions cleanups can vary a lot, but ...
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2
votes
Total reads aligning to each reference within a bam file
This one-liner below will work better for long reads than samtools flagstat in that it only counts the primary alignment for each read and ...
- 3,141
2
votes
Using ONT MinION, why is that we cannot get a full length DNA read?
The MinION should be able to sequence DNA fragments entirely, regardless of the size, if you can get them to the pore intact. That is the problem: during DNA extraction and library preparation you ...
- 1,314
2
votes
Accepted
Error correction within the long read
Yes, there is a tool to do this called R2C2 by the Vollmers lab at UC Santa Cruz.
https://www.biorxiv.org/content/early/2018/06/04/338020
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2
votes
Microbial Classification with 16S Nanopore Data
I would second the answer gringer gave recommending Kraken2. I would suggest using bracken to process the results from Kraken2 if you want to estimate species abundance (the raw Kraken2 results ...
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