49
votes
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Why does the FASTA sequence for coronavirus look like DNA, not RNA?
That is the correct sequence for 2019-nCov. Coronavirus is of course an RNA virus and in fact, to my knowledge, every RNA virus in Genbank is present as cDNA (AGCT, i.e. thydmine) and not RNA (AGCU, i....
M__♦
- 11.2k
31
votes
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Why do some assemblers require an odd-length kmer for the construction of de Bruijn graphs?
From the manual of Velvet:
it must be an odd number, to avoid palindromes. If you put in an even
number, Velvet will just decrement it and proceed.
the palindromes in biology are defined as ...
- 5,497
30
votes
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Why sequence the human genome at 30x coverage?
The earliest mention of the 30x paradigm I could find is in the original Illumina whole-genome sequencing paper: Bentley, 2008. Specifically, in Figure 5, they show that most SNPs have been found, and ...
- 530
24
votes
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Is it possible for coronavirus or SARS to be synthetic?
The scenarios are impossible and would be laughable if they were not so serious. The evidence is in the phylogenetic trees. Its a bit like a crime scene when the forensics team investigate. We've done ...
M__♦
- 11.2k
16
votes
Accepted
Why does this human bam file only have one copy of each chromosome?
The maternal and paternal copies of a chromosome are called haplotypes. Many metazoans (animals) are diploid and have maternal and paternal chromosome contribution during sexual reproduction, not just ...
- 3,141
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 ...
16
votes
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15
votes
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How can we distinguish between true zero and dropout-zero counts in single-cell RNA-seq?
Actually this is one of the main problems you have when analyzing scRNA-seq data, and there is no established method for dealing with this. Different (dedicated) algorithms deal with it in different ...
- 349
14
votes
Why do some assemblers require an odd-length kmer for the construction of de Bruijn graphs?
To expand on the answer above, in case it isn't clear, we show:
Why palindromic sequences must be even in length
Why palindromic sequences induce self-loops in a de Bruijn graph
Why self loops in a ...
- 243
14
votes
Accepted
Fast way to count number of reads and number of bases in a fastq file?
It's difficult to get this to go massively quicker I think - as with this question working with large gzipped FASTQ files is mostly IO-bound. We could instead focus on making sure we are getting the ...
- 861
12
votes
QC measures for NGS sequencing
MultiQC can merge all your different reports into a single one.
Which could be useful once you manage to know which QC tools to use.
- 129
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 ...
- 1,284
12
votes
Accepted
How do PCR duplicates arise and why is it important to remove them for NGS analysis?
In any scenario where depth of coverage is an important factor, PCR duplicates erroneously inflate the coverage and, if not removed, can give the illusion of high confidence when it is not really ...
- 5,060
11
votes
Accepted
What is deep sequencing?
I found a post useful for this topic.
It explains the difference of coverage and depth. It also has a useful explanation on how to calculate coverage and depth.
Here is a copy of what the link says ...
- 226
11
votes
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PCA vs tSNE in single cell RNA-seq
tSNE often offers better visual representation (separation) on such complicated data than PCA. As Micheal pointed out, computing a tSNE embedding over 20.000 gene dimensions is computationally ...
- 643
11
votes
Accepted
A new paper suggests the Corona Virus has "Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1" - What does this mean?
UPDATE: The article has now been withdrawn with the following note:
This paper has been withdrawn by its authors. They intend to revise it
in response to comments received from the research ...
- 3,928
10
votes
Accepted
How to count the number of mapped read in 100-bp window from a BAM/SAM file
one-liner
Here's a gritty one-liner to count the number of reads in a region if you have just one region that you want to investigate. Change the 1 in ...
- 3,141
9
votes
Designing a lab NGS file database schema
The Global Alliance for Genomics and Health has been working on the issue of representing sequencing data and metadata for storage and sharing for quite some time, though with mixed results. They do ...
- 429
9
votes
What is deep sequencing?
There are several questions in your post I'll try to answer each one:
Is there any way to calculate how deep the sequencing is ?
See gringer's answer. TLDR: The depth of the sequencing is how many ...
- 4,692
9
votes
Accepted
What is the index fastq file (sample_I*.fastq.gz) generated when demultiplexing Illumina paired-end runs?
tldr - The I*.fastq.gz file contains the read index sequences.
long explanation
Illumina uses a program called bcl2fastq to demultiplex sequencing runs.
This ...
- 3,141
9
votes
Delete all 4 lines of a fastq read from a fastq file using read ID
Don't do it: Your FASTQ file is either malformed or your FASTQ record spans more than four lines, which is allowed in FASTQ. For a detailed description of what can go wrong in FASTQ parsing see for ...
- 2,206
8
votes
Accepted
Is it possible to perform MinION sequencing offline?
Short answer: yes, but you need to get permission (and modified software) from ONT before doing that.
... but that doesn't tell the whole story. This question has the potential to be very confusing, ...
- 13.5k
8
votes
What is deep sequencing?
Sequencing depth is typically calculated as the number of total bases sequenced divided by the number of bases in the target genome. An Illumina sequencing run with 2x125 bp reads and 500 million read ...
- 13.5k
8
votes
Accepted
How can I count the number of reads that support a variant in a bam file?
If you don't mind a bit of manual counting, then samtools mpileup -f reference.fa -r chr22:425236-425236 alignments.bam will produce output where you can count the ...
- 19.5k
8
votes
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Why do I get so many insertions from Minimap2 on my Nanopore WGS?
Insertions and deletions are the dominant error mode of long read sequencing, including nanopore sequencing. What you see is not unexpected. Things may have improved by now if you would download the ...
- 1,314
8
votes
Accepted
Why don't all reads have adaptors?
Things like this might depend on your specific library prep, but in general: sequencing starts at the end of the adapter, not before it. You will only see adapters if you sequence through the entire ...
- 1,314
7
votes
How to make a distinction between the "classical" de Bruijn graph and the one described in NGS papers?
Several papers have made this distinction, and a few indeed use different terms to distinguish between them. For example, Kazaux et al. (2016) acknowledge that:
These constraints favour the use of ...
- 669
7
votes
How to simulate NGS reads, controlling sequence coverage?
I am working on a Illumina sequencing simulator for metagenomics: InSilicoSeq
It is still in alpha release and very experimental, but given a multi-fasta and an abundance file, it will generate reads ...
- 439
7
votes
QC measures for NGS sequencing
We routinely run both FastQC and FastQ Screen on all of our raw sequencing reads. FastQ Screen is a tool for detecting cross-species contamination. MGA is another similar tool.
There are then lots of ...
- 291
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