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 ...
12
votes
Accepted
Which quality score encoding does PacBio use?
PacBio does use PHRED 33, but it turns out the question may be irrelevant for the newer PacBio Sequel Sequencer, because it reports all base qualities as PHRED 0 (ASCII ...
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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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5
votes
Accepted
estimate genome size: kmer-based approach from PacBio reads
I don't think there is a method that would estimate a genome size using raw long reads.
The genome size estimates based on raw reads are done by fitting a model to kmer spectra (for instance ...
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5
votes
Accepted
Why is a PacBio read length larger than the aligned reference region?
Q1
Probably not - this is probably just displaying the contents of the input (I presume a bam file) exactly as they are encoded.
Q2
The splicing looks a little ...
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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 ...
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4
votes
Accepted
What is the most accurate approach for de Novo sequencing?
HiFi reads can resolve most repeats in a genome. Illumina reads from these repetitive regions are likely to get mismapped and these mapping errors may become consensus errors. Most short-read ...
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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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3
votes
Accepted
Best way to "fish" long reads that match a query sequence
I have seen the blasr aligner typically used for aligning PacBio reads to a reference. Is there a reason why blasr would not be a good choice? I guess the very short sequence you are trying to align ...
- 1,012
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__♦
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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.
...
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3
votes
Accepted
Can pair end reads with high heterozygosity be used to polish PacBio assembly?
Resequencing your sample is not going to make it any less heterozygous. I am assuming that you are working with a diploid organism? Please update your question and specify the genome size and ploidy ...
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3
votes
Accepted
How to install blasr on Mac OS X?
The path of least resistance is probably to run the software inside a container. If you install docker, you can just do the following:
...
- 1,816
3
votes
Accepted
How to subset an SRA file for a single chromosome?
After dumping out the reads using fasterq-dump, you'll need to align them first and then extract those that map to your region of interest. I think minimap2 is an ...
- 3,024
2
votes
How to install blasr on Mac OS X?
I've had many issues with installing blasr on my own, but it's a breeze using (bio)conda, see https://anaconda.org/bioconda/blasr
conda install -c bioconda blasr
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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 ...
- 1,284
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
Accepted
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
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.
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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. ...
- 2,206
2
votes
What assembler is appropriate for High-Fidelity PacBio reads
From the group of Heng Li there is hifiasm.
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2
votes
Accepted
How do you set the coverage in PacBio's Sequel II?
They just want to know the ploidy of the sample. In a diploid sample, e.g. humans, there are two haplotypes. This is mostly important for haplotype resolution (i.e. get the two full genomes in a ...
- 3,649
2
votes
Accepted
How can Iso-Seq reverse transcriptase artifacts be avoided?
It is possible to filter out iso-seq reads with RT artifacts, though I don't know how effective these informatics approaches are. With nanopore, you will get more false junctions in alignment or ...
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2
votes
regex: samtools command to "refine" PacBio IsoSeq data?
The samtools view command you've provided just uses a filter expression to discard records that do not meet the specified expression criteria. In this case, the ...
- 3,024
1
vote
Accepted
What is a PacBio "movie file"?
[disclaimer: not a PacBio person, I'm just vaguely aware of the technology]
Movie files are the PacBio raw data format, representing observed signal intensities in each sequencing well over the course ...
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1
vote
Accepted
Is it possible to filter contaminated reads for raw PacBio sequences (not HiFi reads) before assembly?
I don't think that in principle the issue is any different for PacBio and Illumina; it's just that the process might be noisier with higher error rates. Note that PacBio errors aren't randomly ...
- 3,649
1
vote
Gap-fill assembly with PacBio reads
My understanding is that PBJelly is the tool intended to solve this problem.
This person seems to have used it starting with Illumina scaffolds. I have not personally used it but have heard that it ...
- 3,649
1
vote
polishing assembled genome to QV50 value
15 times is too many times. If you were going to get quiver to get you good QVs it would have happened after 2-3 rounds probably.
Some questions:
Why are you using quiver? Are your reads from RSII ...
- 3,649
1
vote
What assembler is appropriate for High-Fidelity PacBio reads
The HiFi de novo assembly tool that PacBio has been developing is called IPA.
It’s available on pbbioconda for command line use, and as a push-button pipeline in their SMRT Link software.
I looked at ...
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1
vote
How many reads do I need for hybrid assembly
I have never used dbg2olc. However other OLC assemblers managed to do a solid assembly from 20x of corrected long reads. But obviously, the more the better.
In you case of allotetraploid, you need to ...
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