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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Nanopore Flongle vs MinION
Ho 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 ...
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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 ...
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aligner for 1D^2 oxford nanopore data
I am wondering if anyone has tried aligning 1d^2 RNA-seq data. I am trying that on minimap2. It is giving me very low ~50% of mapped reads. it gives ~95% mapped reads for 1D.
I am using the following ...
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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 ...
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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.
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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
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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 ...
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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 ...
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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 30 mouse cell lines, and are deciding between Illumina and Nanopore ...
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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 ...
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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 ...
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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 ...
M__♦
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Extract nanopore read ID & start times from fastq file
I have a fastq file from minION (albacore) that contains information on the read ID and the start time of the read. I want to extract these two bits of information into a single csv file.
I've been ...
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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 ...
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How to simulate nanopore reads?
I have looked already here: Tools for simulating Oxford Nanopore reads
. This doesn't answer my question, because it lists a few Nanopore read simulators, but I have specific problems with each of ...
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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 ...
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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 ...
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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 ...
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How can I improve a long-read assembly with a repetitive genome?
I'm currently trying to assembly a genome from a rodent parasite, Nippostrongylus brasiliensis. This genome does have an existing reference genome, but it is highly fragmented. Here are some ...
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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 ...
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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 ...
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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 ...
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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-...
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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:
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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" ...
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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
...
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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?
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Split FASTQ and matching BAM into matching chunks
I am running a slow downstream analysis on a large set of nanopore reads (approx 3 million), and would like to split them into smaller chunks, run the analysis in massively parallel, and then ...
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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 ...
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Read length distribution from FASTA file
I have a single ~10GB FASTA file generated from an Oxford Nanopore Technologies' MinION run, with >1M reads of mean length ~8Kb. How can I quickly and efficiently calculate the distribution of read ...
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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 ...
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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 ...
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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.
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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 ...
CommunityBot
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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 ...
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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 ...
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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 ...
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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 ...
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wtdbg2: practical implications of k-mer fsize and psize choice
I am using wtdbg2 2.3 to assemble a human genome (sequenced on PromethION from a cell line). I filtered out reads with low average quality, and now I am trying to determine the parameters that will ...
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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 ...
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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 ...
M__♦
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Finding the location and unit length of repetitive sequences within a long read
After discovering a few difficulties with genome assembly, I've taken an interest in finding and categorising repetitive DNA sequences, such as this one from Nippostrongylus brasiliensis [each base is ...
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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 ...
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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 ...
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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.
...
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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 ...
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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 ...
CommunityBot
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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 ...
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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 ...
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Demultiplex nanopore reads with custom barcodes
We have a problem trying to demultiplex MinION sequences with custom barcodes. Do you have any software recommendations we can try for demultiplexing or how to demultiplex these custom barcodes with ...
