15 votes
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Why Illumina if PacBio provides longer and better reads?

There are so many reasons why one might want to prefer Illumina over PacBio (also note that it's a false dichotomy, at least Oxford Nanopore is a competitive sequencing platform): The first (IMHO and ...
6 votes
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Can I stop my nanopore sequencing run if there are no more reads being produced?

Yes, you can click "stop acquisition" and your run won't be negatively affected. All of the reads are saved as they are generated. I am not sure how this will impact live basecalling though if that is ...
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  • 3,061
6 votes
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Coverage calculation: long reads (RNA-seq)

The read length is irrelevant when calculating the mean coverage statistic. It's simply the total number of bases sequenced divided by the target Xome length. In the example provided in the question, ...
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6 votes
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How can I improve a long-read assembly with a repetitive genome?

You can't resolve 20kb near identical repeats/segdups with 10kb reads. All you can do is to bet your luck on a few excessively long reads spanning some units by chance. For divergent copies, it is ...
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  • 5,645
6 votes
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How can I use Nanopore reads to close gaps or resolve repeats in a short-read assembly?

You might want to look into Unicycler (manuscript with more information can be found here); even though it is supposed to be used with bacterial genomes only, it might work well with a small genome ...
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5 votes
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Detecting structural variants with MinION data

There was a Structural Variant breakout session at the London Calling conference this year. Unfortunately I didn't attend that session, but MinION community members have access to Constance Donnell's ...
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5 votes
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How to simulate nanopore reads?

It is important to train an error model as NanoSim does, as we do not fully understand the error processes involved in both the nanopore sequencing process and the basecalling process. Any sort of ...
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  • 3,061
5 votes

Why Illumina if PacBio provides longer and better reads?

Many analyses performed on Illumina machines these days require large numbers of reads. For example, most analyses in ChIP-seq, RNA-seq, ATAC-seq etc, need 10s or even 100s of millions of reads for ...
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  • 3,201
5 votes

Why Illumina if PacBio provides longer and better reads?

Three reasons for Illumina: ...
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  • 7,062
5 votes
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PacBio long-reads impact in transcriptome de novo assembly?

A few comments: Never use N50 as a metric especially for transcriptomes. It has some semblance of relevance for genome assembly, but all that is void for a transcriptome with inherently dynamic ...
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4 votes

How to deal with heterozygosity during polishing of genome assembly based on long reads?

A few possibilities: Falcon Try falcon and falcon-unzip. These are designed exactly for your problem and your data: https://github.com/PacificBiosciences/FALCON Not Falcon If you think you have ...
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4 votes

Questions regarding Nanopore sequencing analysis

1. The quickest way to know if it's a single or multi fast5 is the file name. If it mentions channels and read numbers (e.g. ..._read_9_ch_471_..., then it's a ...
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4 votes
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BED file from .bam alignment structure

I cannot access the pipeline from nanopore, but looking at the python script you provided: ...
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  • 1,638
4 votes
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Difference between paired-end, mate-pair and long read

They are all very different in separate regards, but they all refer to different wet-lab and sequencing protocols/technologies. First, PE (paired end) reads are typically short (50-300) reads, most ...
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3 votes
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Mapping Reads to Known Gene Paralogs with Long Read Technology

Question 1 This command will get you all of supplementary alignments for the reads. This isn't exactly what you want though. You want all of the reads that have more than one mapping. ...
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  • 3,061
3 votes
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How to map short sequences to long reads, recovering all multiply-mapped high-quality matches

LAST has given the best results for me when I've tried to do this, although I agree with @user172818 that it's not a good idea to map really short reads. This is due to a combination of natural ...
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3 votes
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How many reads has my sequencing run produced on minion?

That's indeed the number of reads and that's quite low. How was your pore occupancy (number of pores sequencing) and your flow cell QC (number of pores good enough for sequencing)? How was your ...
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3 votes

How to deal with heterozygosity during polishing of genome assembly based on long reads?

You could also have a go at Canu. It's designed for long-read assembly (both PacBio and Nanopore), although not specifically for complex population sequencing. It tries to strip a genome down into its ...
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3 votes
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Is nanopolish worth it since faster polishing software is available?

Nanopolish is necessary if you want to get high-quality consensus. racon etc don't use signal data. They can't achieve high quality. pilon at times doesn't work well. Even if it worked perfectly, it ...
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3 votes

What assembler is appropriate for High-Fidelity PacBio reads

As recently tweeted by A. Phillippy, Canu v1.9 now supports HiFi reads. Therefore you should just read their manual and look for the implementation :)
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2 votes
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Finding the location and unit length of repetitive sequences within a long read

I've sorted the visualisation out. Here are three alternative representations of repetitive structures for the same sequence: These were generated using the same R script, callable from the command ...
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2 votes

Finding the location and unit length of repetitive sequences within a long read

It could be an idea to fragment the long reads into small sequences, like simulating Illumina reads of 150 bp, and then map these small sequences against the original long reads and extract regions ...
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2 votes

Structural variant calling for low-coverage PacBio data

There is an evaluation of PB Honey and Sniffles algorithms for low coverage PacBio datasets in this preprint and another evaluation is shown on this poster. Both reports agree that optimal is (...
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2 votes
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What to do with the configuration test cell after configuring the MinION with it?

I would keep it, in case you need to do another configuration later on. I have used mine for that purpose. I think storage doesn't really matter, I just put it in a drawer somewhere. Keep it away ...
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2 votes
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Does MinKNOW work with Mac OSX high sierra 10.13.1?

Some replies from ONT is: OS X is supported and you can view the installation instructions on our Downloads page linked below. At the moment High Sierra is not officially supported as we have ...
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2 votes
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Scaffolding a genome with hybrid data

Try LR_Gapcloser. I've used L_RNA_Scaffolder for trying to scaffold a genome (which turned out to be more complex than I had expected). It looks like LR_Gapcloser (written by the same people) is ...
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2 votes
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Can I pilon-polish long reads with Illumina short reads to improve structural variant detection?

I'd recommend using Canu for correction rather than Pilon, because it has a component that is specifically designed for read-level correction. The newest version of Canu (v1.7) will track all reads ...
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2 votes
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How to interpret the SMRT Link base modification algorithm output?

It sounds like you're looking for the modifications.csv file, rather than the modifications.gff file. The ...
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2 votes
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calling diploid SNVs from long reads

You may want to try the longshot tool (https://github.com/pjedge/longshot) developed for calling variants in diploid genomes from long read data.
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2 votes

calling diploid SNVs from long reads

You could try aligning your reads to the draft reference genome with for example minimap2 and calling variants with freebayes. ...
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