20
votes
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Read length distribution from FASTA file
If you want something quick and dirty you could rapidly index the FASTA with samtools faidx and then put the lengths column through R (other languages are available)...
- 772
12
votes
Read length distribution from FASTA file
Statistics for nanopore reads are tricky because of the huge range of read lengths that can be present in a single run. I have found that the best way to display lengths is by using a log scale on ...
- 13.8k
11
votes
Accepted
Tools for simulating Oxford Nanopore reads
Simulators designed specifically for Oxford Nanopore:
NanoSim
NanoSim-H
SiLiCO
ReadSim
DeepSimulator
General long read simulators:
Loresim
Loresim 2
FASTQsim
LongISLND
For an exhaustive list of ...
- 1,899
9
votes
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Extract nanopore read ID & start times from fastq file
awk '{if(NR%4==1) print $1, $5}' file.fastq | sed -e "s/ start_time=/, /" -e "s/^@//"
The awk command gets the first of every ...
- 19.6k
9
votes
Tools for simulating Oxford Nanopore reads
By chance, just today I've heard of a nanopore read simulator, NanoSim. It is released under a GPL license. I have never used it, though...
- 669
8
votes
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Building STAR Genome Index for nanopore RNA sequencing
The parameter is used to determine how much sequence STAR indexes on each side of a splice junction to improve its alignment accuracy. For very long reads, this may not be ideal. I am not sure if ...
- 752
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 ...
- 3,301
8
votes
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Why do I get so many insertions from Minimap2 on my Nanopore WGS?
Insertions and deletions are the dominant error mode of long read sequencing, including nanopore sequencing. What you see is not unexpected. Things may have improved by now if you would download the ...
- 1,314
7
votes
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How can I improve a long-read assembly with a repetitive genome?
"A few" 100kb reads won't help much. You need to apply the ultra-long protocol, which is different from the standard protocol.
You can't resolve 20kb near identical repeats/segdups with 10kb ...
- 6,485
7
votes
Tools for simulating Oxford Nanopore reads
In addition to the already mentioned NanoSim, there is also SiLiCO and ReadSim (although it hasn't been updated in over 2 years, so I am not sure how relevant it is at this point considering how fast ...
- 2,169
7
votes
Read length distribution from FASTA file
Using Biopython and matplotlib would seem like the way to go, indeed.
It really just boils down to three lines of code to get that graph:
...
- 179
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 ...
- 13.8k
6
votes
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Compare alignment quality of multiple sequencing runs aligned against the same reference genome
Qualimap will do this for you.
Go to qualimap.bioinfo.cipf.es
Run qualimap (default params are fine) on each BAM file
Open up the HTML output, and you can read off the %identity (they measure the ...
- 952
6
votes
Extract nanopore read ID & start times from fastq file
Since the string start_time will only appear on the header line, or else you don't have a valid fastq file, you can simply do:
...
- 9,662
6
votes
Accepted
How can I use Nanopore reads to close gaps or resolve repeats in a short-read assembly?
You might want to look into Unicycler (manuscript with more information can be found here); even though it is supposed to be used with bacterial genomes only, it might work well with a small genome ...
- 967
6
votes
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How to convert fastq to fast5
NOTICE:
I have altered my answer slightly from the original as I have turned the original script into a pip installable program (with tests) and have updated the ...
- 663
6
votes
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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 ...
- 782
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 ...
- 3,141
6
votes
Significance and timing of "mux scans"
Yes, your understanding is largely correct. This originates from the situation that for each detector on a nanopore array there are 4 pores. I'll explain mux scans and groups, but this is outdated ...
- 1,314
6
votes
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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 ...
- 277
6
votes
How much does Nanopore cDNA Sequencing Cost?
The standard PCR-cDNA barcoding kit is \$750 USD, and has enough reagents for six runs, with each run using up to 24 samples (i.e. 144 samples total). Each run also needs one MinION flow cell (\$900 ...
- 13.8k
5
votes
Visualisation of long read RNA-Seq splicing
DNASTAR's software is for purchase, but high quality. GenVision Pro does genomic visualization, including Sashimi plots.
Edit: not sure why this answer is being downvoted, unless it's because the ...
5
votes
How do you generate read-length vs read-quality plot for long-read sequencing data (e.g., MinION)?
I also wrote a package to create various plots from Oxford Nanopore sequencing data and alignments: NanoPlot. It can be installed through pip (see also the README on Github). In addition to multiple ...
- 1,314
5
votes
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Detecting structural variants with MinION data
There was a Structural Variant breakout session at the London Calling conference this year. Unfortunately I didn't attend that session, but MinION community members have access to Constance Donnell's ...
- 13.8k
5
votes
Read length distribution from FASTA file
There are several potential approaches. For example:
histogram of sequence lengths in the Biopython tutorial
plot_distribution from the Ruby-based biopieces framework
various solutions to get ...
- 646
5
votes
Read length distribution from FASTA file
You specifically asked about FASTA files, but it's important to always consider read length and quality jointly when assessing high-error long-read data. FASTA files do not provide the quality. This ...
- 1,304
5
votes
Accepted
Split FASTQ and matching BAM into matching chunks
Hmm, it's hard to think of a super efficient way of doing this (assuming the files aren't ordered the same - if they are then this whole answer is basically redundant). And also assuming the read ids ...
- 663
5
votes
Accepted
STAR-long parameters for aligning RNA ONT reads to genome
I've had great results using minimap2, particularly when combined with a pre-treatment of Canu for error correction (using minimap2 for the read-to-read mapping):
...
- 13.8k
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
How to simulate nanopore reads?
It is important to train an error model as NanoSim does, as we do not fully understand the error processes involved in both the nanopore sequencing process and the basecalling process. Any sort of ...
- 3,141
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