Questions tagged [nanopore]

Questions specific to nanopore sequencing. For general question about long reads, use tag long-reads instead and for questions about specific sequencer use a specific sequencer tag (i.e. minion, gridion)

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single_to_multi_fast5 does not collect all the single files if the input folder contains mixed types of fast5 files

I have a dataset that contains thousands of mixed multiple and single fast5 files in a non-homogenous folder structure. The reads and fast5 files are coming from different labs and some labs gave ...
Marjan's user avatar
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Nanopore Flongle vs MinION

How do the Oxford Nanopore Flongle flow cells perform compared to the standard MinION flow cells, when it comes to reliability and error rates? I found this comparison article from last year, from ...
Joel's user avatar
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How should I use dorado basecaller to calculate translocation time? Can I change models' config files?

I am trying to use dorado basecaller to process ONT data. I need to make the move table and I am doing it with --emit-move. ...
Marjan's user avatar
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Is my reference sequence too small?

I'm trying to map ONT long reads to a portion of a gene I'm looking at. The region is about 25bp long. When I search for the region in the document it pulls up the sequence in every read but when I ...
rimo's user avatar
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Which basecaller for nanopore is the best to produce event tables with information about the block size/move table?

I previously used albacore version 2.3.1 to make initial move tables, but then I re-squiggle using Tombo version 1.5.1 to fix the errors. Example of move table produced by albacore, in which all the ...
Marjan's user avatar
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How do I measure the proportion of different bacteria in a sample from a high-throughput sequencer?

[this question is based on a question that was asked on Reddit] I have sequenced some [mostly cell-free] DNA from a sputum sample on a nanopore sequencing machine, and would like to know what is in it ...
gringer's user avatar
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How much does Nanopore cDNA Sequencing Cost?

[this question is based on a question that was asked on Reddit] We're interested in doing whole-transcriptome cDNA sequencing of 24 mouse cell lines, and are deciding between Illumina and Nanopore ...
gringer's user avatar
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Improving prokaryotic assembly with other contig/scaffold-level data?

I have what at first sight appears to be a high-quality MAG (~10 pieces, high completion%) that I built from a hybrid assembly (Illumina + Nanopore data) from a cyanobacterium. Workflow: Quality ...
Laura's user avatar
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1 answer
355 views

Reading a Fast5-file with Python

I am trying to extract data from fast5-file with python 3.9.13 in Ubuntu. I have found a library "fast5_research"(This package comprises an API to HDF containers used by the research groups ...
Shwarz's user avatar
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Coordinate numbering between forward (+) and reverse (-) strand in Tombo

My question is related to the output of Tombo re-squiggling algorithm on nanopore data. Given the position/coordinate on one strand (+ or -), I want to find the coordinate of its exact location on the ...
Marjan's user avatar
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How can Iso-Seq reverse transcriptase artifacts be avoided?

My end goal is annotate a de-novo assembled genome. When trying to select the best method for transcriptome assembly I read Iso-seq was the preferred method. However other people suggested Nanopore as ...
Caterina's user avatar
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1 answer
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Get number of reads with a single, (almost) exact match to the full length of a reference sequence

I can't find an answer to this in previous questions, so hoping someone can help me now. We have nanopore sequenced a PCR product, and I've filtered our reads to +/- 10 bp of the expected product ...
Nat R McB's user avatar
2 votes
1 answer
303 views

How can I generate ASV file from nanopore sequencing data?

I am converting the fast5 file to fastq by using guppy basecaller after that by using kraken2 classified the sequence. Now I am trying to generate ASV file. Is this possible to generate ASV file from ...
twinkle's user avatar
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What is the contributing factor in single measurements of nanopore sequencing? One base, a k-mer or difference between the base left and entered?

My question is about nanopore sequencing and specifically about the current that is measured by the device in each measurement. The question is: In each measurement in nanopore sequencing, the change ...
Marjan's user avatar
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somatic SNV tool for ONT samples

I'm looking for a tool to call somatic single nucleotide variations on reads from an Oxford Nanopore Technologies (ONT) run. I have both tumor and normal samples and have already aligned them to a ...
Walter Gallego Gómez's user avatar
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1 answer
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Tree Building Algorithm that treats gaps as deletions

I'm part of a nanopore sequencing experiment that will sequence several generations of viruses. The intent is to perform directed evolution by putting selective pressure on these viruses and tracking ...
CCranney's user avatar
4 votes
1 answer
542 views

How can I get the duration of the events in Nanopore?

PacBio has the concept of IPD (InterPulse Duration) which is the time between two detected consecutive sequences in the raw signal. I have been trying to extract this value in the Nanopore, but I ...
Marjan's user avatar
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3 votes
2 answers
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Understanding fast5 format

I would like to reproduce the results on figure 1 from THIS publication. I downloaded the data and explore it with h5ls and h5dump based on HDF5. I would like to understand: ...
aerijman's user avatar
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2 answers
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Nanopore variant calling

So far I haven't done any variant calling as such. Nanopore I have used for 16s microbiome data. Now My question/doubt so how do I proceed for nano-pore virus sequencing data Steps: I get fast5 files ...
kcm's user avatar
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1 vote
3 answers
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Polishing PacBio or ONT with Illumina

Which tool would be good to polish PacBio or ONT with Illumina? Thank you in advance
user977828's user avatar
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How do I tell that a MinION run has finished based on the files produced?

I want to automate some scripts which should run after a MinION run completes. Is there a file which is produced by MinKNOW which will enable me to tell that the run has finished?
flashton's user avatar
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Doing functional enrichment of microbiome data

I have done gene set enrichment analysis which is now straight forward. Such as taking gene set and making a query. For microbiome data it is not that straight forward which I found out. Step done so ...
kcm's user avatar
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What are some ways to error correct Oxford Nanopore long read sequencing?

I am sequencing long read genomic sequences to assemble MHC region haplotypes in a non model organism using Cas9 Sequencing Kit (SQK-CS9109) using flongle adaptors in the minION from Oxford Nanopore ...
mk894's user avatar
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Detecting SARS-CoV-2 variants from the mixed virus population

I have a fastq file from Nanopore sequencing data that contains reads from both the UK and South Africa variants of SARS-CoV-2. The variants are identified by three key mutations in the receptor-...
L R Joshi's user avatar
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1 answer
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Oxford nanopore promethion; does guppy3 separate fast5 into pass and fail? If not what does?

just a quick question about fast5 pass fail folders. I have been working under the assumption that the fast5 pass fail folders I was given as raw data from our ONT vendor came from them doing base ...
Liam McIntyre's user avatar
1 vote
1 answer
1k views

Upgrading guppy basecaller on Oxford Nanopore Gridion via PPA

I am trying to update the guppy_basecaller (current version installed is 3.2.10+aabd4ec) on a ONT Gridion device. I am initially trying to update it via the ...
BCArg's user avatar
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1 answer
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Nanopore data clarification regarding merging of samples

We had same set of sample ran on nanopore platform twice. The issue on hand is. First run was done it was nearly done but the power went off. Second run was done completely with enough sequencing ...
kcm's user avatar
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ambiguous calls in nanopolish

I am converting scores into frequencies (from nanopolish results) using Nanopore scripts. The script filters out lots of rows on the premise that reads where ...
aerijman's user avatar
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4 votes
2 answers
76 views

What is the typical host-to-bug DNA ratio found in nanopore sequencing without amplification?

I'm interested in sequencing a human sputum sample using an ONT MinION without performing any type of whole genome DNA amplification or targeted PCR. Has anyone found a good reference (or anecdotal ...
TimD1's user avatar
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Master of Pores Installation Mac OS Mojave 10.14

I'm bit of a newbie with ONT analyses and am having some trouble with the Master Of Pores installation instructions (https://biocorecrg.github.io/master_of_pores/install.html). I've managed to install ...
guray00's user avatar
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1 vote
3 answers
447 views

Recommended workflow for RNA long read Kallisto-like TPM estimation?

On short reads, Kallisto/Salmon is a standard workflow for measuring RNA transcript counts in TPM. However, when I tried to Google a similar workflow for long reads, it's not clear there is a ...
SmallChess's user avatar
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nanopore QC measures on fastq file

I am working with virus nanopore sequencing data. I have to remove host genome from the reads. I want to run the QC on the fastq file after host genome subtraction. I have used nanoplot. It is slow. I ...
Arun Venugopalan's user avatar
1 vote
0 answers
39 views

Nanopore microbiome data analysis 16s rRNA data

The steps I have done so far: used guppy to do base calling from fast5 to fastq. In each barcode file I have 1960 fastq files which I have merged. trimmed the fastq files using NanoFilt Now what I ...
kcm's user avatar
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0 votes
1 answer
459 views

snakemake - using output file not explicitly declared as input for subsequent rule

I am writing a snakemake that, up to now should use raw sequencing reads to produce a draft assembly using canu use minimap2 to align the raw reads against the draft assembly, generated by canu. ...
BCArg's user avatar
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1 vote
0 answers
95 views

Train a new basecalling model for nanopore?

Basecalling on nanopore requires a machine learning neural network model, for example here A popular software from Nanopore for it is https://github.com/nanoporetech/taiyaki, which has many people ...
SmallChess's user avatar
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0 votes
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Program Quality control for data sequencing (nanopore)

Hope all of you are fine. I am wondering if exists a program easy to use that can bring me Basic Statistics of Quality Control from raw data made by MinION device (nanopore). I have read about ...
GrayC's user avatar
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0 votes
1 answer
348 views

Kallisto for Nanopore?

Although the Kallisto software was designed for RNA, it has also been very reliable in counting selected FASTA sequences in DNA sequencing. For example, given a WGS sample and my FASTA file. Kallisto ...
SmallChess's user avatar
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0 votes
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nanopore - where to retrieve information from the basecaller used

Where can I find information about the basecaller used in a specific nanopore run (we have a Gridion machine)? I know already that we have Guppy v. 3.0.3, which I ...
BCArg's user avatar
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1 vote
1 answer
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How do you map nanopore fast5 files?

Is there a best practices for mapping Oxford Nanopore files to a reference? Is there a tool that can take a tarball of fast5 files and map them directly or do they need to be converted to fastq first?...
juniper-'s user avatar
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3 votes
2 answers
441 views

Microbial Classification with 16S Nanopore Data

I have recently been tasked with analyzing a heterogeneous microbial sample composed of 16S data generated on a MinION sequencer, with the goal of assigning taxonomies and calculating respective ...
Sparkflyer's user avatar
2 votes
1 answer
436 views

BED file from .bam alignment structure

I am using a pipeline from nanopore to detect structural variants (SVs) in a human sample with long-reads sequencing. The first steps of the pipeline are: index the reference genome with ...
BCArg's user avatar
  • 283
2 votes
1 answer
536 views

Nanopore FASTQ header specifications

Is there a specification anywhere for how a Nanopore FASTQ header should be formatted? I am creating strategies for generating test FASTQ files and would like to ensure I am generating "correct" ...
Michael Hall's user avatar
2 votes
1 answer
688 views

Oxford Nanopore mapping Quality and sequencing error

I'm analyzing some Oxford Nanopore sequencing reads, with several articles stating the sequencing error is between 5% and 15%. Since its 1D and not 1D^2 sequencing i would assume the sequencing error ...
Rasmus Henriksen's user avatar
3 votes
3 answers
182 views

Compare multiple alignment results' aligned bases

I have aligned a nanopore data set to a reference genome with graphmap, minimap2 and BLASR. The alignment results are stored in BAM files. I would like to do some concordance assessment, looking at ...
Danielle Zhang's user avatar
3 votes
2 answers
5k views

Error while calling bcftools mpileup - Failed to open -: unknown file type

I have sequenced a bacterial genome with a GridIon from ONT. Basically what I want to check is whether or not trimming 50 bps at the beginning of the reads will improve alignment against the reference ...
BCArg's user avatar
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6 votes
1 answer
3k views

How does MinKNOW classify 1D reads as "pass" or "fail"?

I have found a couple of sources1,2 that indicate that a read in a 1D² run is classified by MinKNOW as "pass" and put into the fastq_pass folder if both of the ...
Mark Amery's user avatar
2 votes
1 answer
1k views

How do you normalise read coverage in a BAM file?

This is a question from the Oxford Nanopore community, from user Michael Radzieta: I have sequenced some plasmids using the rapid barcoding kit, I have attempted to assemble the data using several ...
gringer's user avatar
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What is a signal 'chunk' in the context of Nanopore sequencing?

Does one chunk correspond to the raw signal of a single read read? Why does messing with this parameter affect basecalling results?
Nick Chadsbury's user avatar
6 votes
1 answer
42 views

Help with 1D^2 library shearing

I've done my 1st trial of 1D^2 whole genome sequencing using LSK-SQK308 kit with R9.5 flowcell. Without doing library fragmentation, I encountered the unexpected library shearing during sequencing. ...
yiyi_Z's user avatar
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3 votes
1 answer
3k views

Significance and timing of "mux scans"

I'm using MinIONQC to do quality control on some ONT data. The software plots several read characteristics over time (hours passed during the sequencing process). These plots contain several vertical ...
Daniel Standage's user avatar