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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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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2answers
62 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 ...
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1answer
39 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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0answers
38 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" ...
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1answer
62 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 ...
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3answers
83 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 ...
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2answers
324 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 ...
3
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1answer
227 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 ...
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1answer
160 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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1answer
37 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?
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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. ...
3
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1answer
453 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 ...
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1answer
235 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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2answers
598 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 ...
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1answer
127 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 ...
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0answers
210 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 ...
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1answer
373 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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1answer
305 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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1answer
143 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....
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1answer
209 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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1answer
64 views

Using ONT MinION, why is that we cannot get a full length DNA read?

Here, https://www.youtube.com/watch?v=E9-Rm5AoZGw, ONT claims they can read non-fragmented DNA and that with the hairpin like structure the double strands can be read at once. Then why is that MinION ...
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3answers
353 views

Total reads aligning to each reference within a bam file

I have two PCR amplicons that have been multiplexed and sequenced using the nanopore minion. I have aligned the fastq reads using minimap2 with a reference file containing both amplicon sequences and ...
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2answers
337 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 ...
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1answer
417 views

Extracting strand specific reads from MinION cDNA-PCR protocol

I recently performed my first MinION run, and now I'm trying to analyze the data. Being pretty new to the bioinformatics field, I was hoping some of you could help me out. As a bit of background, I'...
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2answers
816 views

Can I stop my nanopore sequencing run if there are no more reads being produced?

I am sequencing a whole genome on MinION. I have used this flow cell for 6 hours for the phage lambda control. That gave me around 300,000 reads. I started a 48h sequencing run on the same flow cell ...
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0answers
95 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 ...
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1answer
716 views

How many reads has my sequencing run produced on minion?

I am running a 48 h sequencing protocol on a FLO-MIN107, on MinION, with DNA library-prepped with SQK-RAD004. MinKNOW is installed on my Mac and controlling the MinION. I have found the following ...
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1answer
746 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 ...
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1answer
157 views

Basecalling process carried out by full_1dsq_basecaller.py/.exe

I am unable to understand what exactly the full_1dsq_basecaller.py/.exe script does. Is it giving the consensus sequence for linear and complementary strand, or it is just giving the reads that came ...
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1answer
2k views

Set directory where MinKNOW writes FAST5 files

I would like to set the folder in which MinKNOW writes the raw data. How can I do this? I do not currently know where MinKNOW will output my data. What is the default directory where MinKNOW outputs ...
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2answers
211 views

Can I use my computer while MinION is sequencing without negatively affecting the run?

I have a Mac Book Pro and I am about to start a sequencing run on the MinION. MinKNOW is going to be running for 2 days. Can I use my computer during sequencing? Can I browse the web and use Excel, ...
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1answer
2k views

Difference between 1D and 1D^2 data

I am working on ONT data. Initially, I have worked on data from 1D using Minimap2 aligner. I came across 1D^2 from ONT website. I was wondering how different is the data from two techniques? What is ...
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1answer
256 views

What to do with the configuration test cell after configuring the MinION with it?

What to do with the configuration test cell after configuring the MinION with it? I received a MinION with a configuration test cell in it. After running a configuration in the MinKNOW software while ...
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4answers
4k views

What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?

What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing? I am about to start a MinION run on my laptop. What should I consider when choosing my basecaller? ...
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1answer
497 views

Does MinKNOW work with Mac OSX high sierra 10.13.1?

I have done a compatibility check as described here on my computer to know if I can use MinKNOW on my mac laptop. It says that my OS is incompatible and that the OSX must be Yosemite or El Capitan. ...
3
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1answer
291 views

Removing repeated reads from nanopore 1D² reads

This is summary of discussions and a question that was posted by Devon O'Rourke on Twitter Following Albacore basecalls on a 1D² library I get two sets of .fq files, summary stats, etc.: one for the ...
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3answers
300 views

sort a fasta file containing the Oxford Nanopore Technologies (ONT) header by sequencing start_time ascending

I have basecalled ONT reads and converted them to multifasta. The multifasta contains the original ONT headers in this format: ...
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2answers
229 views

Canu assembly not making a single consensus?

I've downloaded reads from this BioProject. Using canu with default parameters (no correction), I've got 4 contigs, none of which really look like the reference plasmid here. The command I used was: ...
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2answers
720 views

How to get Nanopore MinION fast5 from SRA

I found some Nanopore MinION data on SRA, which I would like to investigate. I use sratoolkit for Illumina data all the time, but I am not sure how to get the ...
6
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1answer
521 views

STAR-long parameters for aligning RNA ONT reads to genome

Are there any suggested parameters to align ONT reads to the reference genome using STAR-long? For now, I used the parameters suggested here, but I noticed a weird behaviour. I have RNA reads (D. ...
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2answers
273 views

Error rate setting in Canu error correction

I want to use Canu to correct my nanopore long read (version: MinION R9.5), but I am not quite sure how to set the correctErrorRate. Should I follow the Canu manual (Nanopore R7 2D and Nanopore R9 1D ...
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4answers
92 views

Introduce errors in reference transcripts according to external dataset error model

I would like to modify some reference transcripts from Ensembl (D. melanogaster) to introduce a controlled rate of random errors in the sequences. The idea would be to introduce random base ...
7
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1answer
237 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 ...
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2answers
1k views

How to convert fastq to fast5

fast5 is a variant of HDF5 the native format in which raw data from Oxford Nanopore MinION are provided. You can easily extract ...
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3answers
519 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 ...
7
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1answer
768 views

How can I use Nanopore reads to close gaps or resolve repeats in a short-read assembly?

Low coverage MinION reads should be useful to close gaps and resolve repeats left by short-read assemblers. However, I haven't had any success with the software I know about. I'm aware of the ...
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2answers
1k 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 ...
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2answers
187 views

How to estimate whether a long-read is meaningful sequence?

The setup Imagine that I work on an organism without a reference genome, and that the closest reference genome I can get is quite diverged. E.g. ~10% diverged in terms of SNVs when measured with ...
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2answers
402 views

Minion channel ID's from Albacore

The latest version of Albacore from Oxford Nanopore Technologies calls bases from raw fast5 files. A useful piece of output is the sequence_summary.txt, which is a ...
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3answers
320 views

Visualisation of long read RNA-Seq splicing

I have a dataset of Oxford Nanopore cDNA reads. Many of my reads are full-length or close to full-length transcripts, and I and am interested in examining alternative splicing. For this, I would like ...