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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1 answer
24 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 ...
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1 vote
1 answer
32 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 ...
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2 votes
1 answer
20 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 ...
1 vote
1 answer
55 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 ...
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4 votes
1 answer
54 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 ...
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2 votes
0 answers
23 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 ...
3 votes
1 answer
46 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 ...
4 votes
1 answer
115 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 ...
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3 votes
2 answers
227 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: ...
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2 votes
2 answers
88 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 ...
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1 vote
3 answers
136 views

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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0 answers
61 views

NanoPlot Error : Kaleido Problem

I'm trying to visualize my fastq file using NanoPlot 1.38.1, but it ends up with this error in the log file: ...
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1 vote
1 answer
69 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?
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1 vote
0 answers
34 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 ...
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1 vote
1 answer
521 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 ...
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2 votes
1 answer
52 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-...
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1 vote
1 answer
662 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
828 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 ...
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0 votes
1 answer
101 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 ...
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0 votes
1 answer
26 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 ...
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3 votes
2 answers
53 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 ...
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1 vote
1 answer
95 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 ...
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1 vote
3 answers
241 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 ...
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0 votes
0 answers
125 views

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 ...
1 vote
0 answers
33 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 ...
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0 votes
1 answer
265 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. ...
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1 vote
0 answers
75 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 ...
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0 votes
2 answers
95 views

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 ...
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-1 votes
1 answer
240 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 ...
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0 votes
1 answer
386 views

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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1 vote
1 answer
92 views

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?...
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3 votes
2 answers
368 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 ...
2 votes
1 answer
245 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 ...
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2 votes
1 answer
231 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
1 answer
350 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 ...
3 votes
3 answers
107 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 ...
3 votes
2 answers
4k 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 ...
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5 votes
1 answer
2k 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 ...
1 vote
1 answer
862 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 ...
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0 votes
1 answer
122 views

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?
6 votes
1 answer
38 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. ...
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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 ...
2 votes
1 answer
406 views

Is nanopolish worth it since faster polishing software is available?

For Oxford Nanopore contigs produced by any long-read assembler has anyone performed any benchmarks to compare the polishing tools racon, ...
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4 votes
2 answers
2k 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 ...
7 votes
1 answer
590 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 ...
7 votes
1 answer
355 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 ...
5 votes
1 answer
807 views

Why do I get so many insertions from Minimap2 on my Nanopore WGS?

I'm a starting my analysis on nanopore whole genome sequencing. I start my analysis from this popular Github. The sample I downloaded was WGS for NA12878, so I would assume it's alignment to GRCh38 ...
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6 votes
1 answer
487 views

Albacore basecalling running but outputs 0 reads

I am trying to basecall data produced by the MinION using the SQK-LSK109 kit and FLO-MIN106 flowcell via the command-line. My version of albacore is the latest (v2.3.4). I tried running using the ...
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4 votes
1 answer
204 views

Question on nanopore sequencing data process pipeline (cDNA-PCR)

I recently started doing the analysis on nanopore sequencing data. As I was searching for some help on pre-processing of the data, I found your nice setup pipeline created here: https://www.protocols....
4 votes
1 answer
237 views

Error correction within the long read

I am going to get some data from plasmid sequencing to identify SNPs on the plasmids. What is done in the lab is the following: The plasmids are purified by size. We amplify the plasmids using the ...
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