16
votes
Accepted
What reasons are there to choose 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 ...
7
votes
Accepted
How can I improve a long-read assembly with a repetitive genome?
"A few" 100kb reads won't help much. You need to apply the ultra-long protocol, which is different from the standard protocol.
You can't resolve 20kb near identical repeats/segdups with 10kb ...
- 6,605
6
votes
Accepted
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 ...
- 3,201
6
votes
Accepted
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
Accepted
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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6
votes
Questions regarding Nanopore sequencing analysis
[Note: As of 2024, ONT now uses pod5 as the standard output format from their MinKNOW sequencing program, and all pod5 files are "multi" pod5 files.]
1. The quickest way to know if it's a ...
- 15.1k
5
votes
Accepted
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
Accepted
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 ...
- 3,201
5
votes
What reasons are there to choose 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 ...
- 3,341
5
votes
Accepted
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 ...
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 ...
- 972
4
votes
Accepted
BED file from .bam alignment structure
I cannot access the pipeline from nanopore, but looking at the python script you provided:
...
- 1,698
4
votes
Accepted
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 ...
4
votes
3
votes
Accepted
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
Accepted
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 ...
- 1,464
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 ...
- 15.1k
3
votes
Accepted
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 ...
- 15.1k
3
votes
What reasons are there to choose Illumina if PacBio provides longer and better reads?
Three reasons for Illumina:
Much better for a large number of samples (easily handle 96 samples).
SNP calling is much better - much greater depth
Hardware costs, an Illumina MiSeq machine is cheap
...
M__♦
- 13k
3
votes
Accepted
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.
...
- 3,201
3
votes
How to simulate nanopore reads?
I could use NanoSim, but that tool seems to be very difficult to use. I only have a reference genome, but you need training data as well?
Karel Břinda's fork, NanoSim-H, ships with pretrained models (...
- 277
3
votes
Accepted
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 ...
- 6,605
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
Accepted
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 ...
- 1,414
2
votes
Accepted
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.
- 36
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. ...
- 2,286
2
votes
Accepted
Error correction within the long read
Yes, there is a tool to do this called R2C2 by the Vollmers lab at UC Santa Cruz.
https://www.biorxiv.org/content/early/2018/06/04/338020
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2
votes
Using ONT MinION, why is that we cannot get a full length DNA read?
The MinION should be able to sequence DNA fragments entirely, regardless of the size, if you can get them to the pore intact. That is the problem: during DNA extraction and library preparation you ...
- 1,464
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 ...
- 141
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 (...
- 5,652
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