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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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50 views

Normalization in Sequence Analysis Research

I am currently engaged in a research project that involves DNA sequence analysis, utilizing nanopore and KMA alignment software (k-mer alignment). As I delve deeper into my research, I am faced with a ...
2 votes
1 answer
450 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 ...
2 votes
1 answer
1k views

Albacore wheel not supported on this platform (Mac OS X El Capitan)

I download the Albacore wheel from Oxford Nanopore. I activated my conda environment that has Python 3 as the default python version. I tried to install Albacore with ...
1 vote
1 answer
31 views

Finding insertion in a plasmid

This question was also asked on Biostars I received a set of Nanopore long reads of a ~3kbp long known plasmid with an unknown insert between 1kbp and 7kbp. Could you suggest a pipeline to assemble ...
10 votes
2 answers
2k views

How do you generate read-length vs read-quality plot for long-read sequencing data (e.g., MinION)?

How do you generate read-length vs read-quality plot (heat map with histograms in the margin) for long-read sequencing data from the Oxford Nanopore Technologies (ONT) MinION? The MinKNOW software ...
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 ...
9 votes
1 answer
1k views

Questions regarding Nanopore sequencing analysis

I am new to Nanopore sequencing analysis. I have a couple of questions regarding it which are as follows: How do I know if my fast5 file is multiread or single read file? Is there a way to combine ...
1 vote
3 answers
590 views

Polishing PacBio or ONT with Illumina

Which tool would be good to polish PacBio or ONT with Illumina? Thank you in advance
2 votes
1 answer
2k 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 ...
2 votes
1 answer
94 views

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 ...
1 vote
1 answer
2k views

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 ...
0 votes
1 answer
110 views

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 ...
6 votes
2 answers
3k views

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 ...
2 votes
1 answer
586 views

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 ...
2 votes
1 answer
166 views

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 ...
1 vote
1 answer
419 views

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. ...
2 votes
1 answer
355 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 ...
3 votes
1 answer
755 views

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 ...
2 votes
1 answer
100 views

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 ...
2 votes
1 answer
279 views

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 ...
2 votes
2 answers
233 views

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 ...
7 votes
3 answers
1k views

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 ...
2 votes
1 answer
114 views

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 ...
5 votes
2 answers
1k views

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 ...
1 vote
0 answers
43 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 ...
1 vote
1 answer
43 views

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 ...
2 votes
1 answer
229 views

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 ...
18 votes
1 answer
652 views

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 ...
4 votes
1 answer
111 views

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 ...
4 votes
1 answer
680 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 ...
4 votes
1 answer
89 views

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 ...
2 votes
1 answer
58 views

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-...
3 votes
2 answers
1k views

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: ...
2 votes
1 answer
621 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" ...
2 votes
2 answers
127 views

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 ...
1 vote
1 answer
259 views

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?
7 votes
1 answer
563 views

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 ...
1 vote
0 answers
43 views

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 ...
28 votes
7 answers
9k views

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 ...
1 vote
1 answer
1k views

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 ...
1 vote
1 answer
110 views

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 ...
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. ...
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 ...
1 vote
3 answers
502 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 ...
1 vote
1 answer
1k views

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 ...
1 vote
1 answer
2k 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 ...
0 votes
1 answer
327 views

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 ...
7 votes
1 answer
380 views

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 ...
0 votes
1 answer
45 views

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 ...
1 vote
0 answers
102 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 ...