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
29
votes
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Uppercase vs lowercase letters in reference genome
What does this soft masking actually mean?
A lot of the sequence in genomes are repetitive. Human genome, for example, has (at least) two-third repetitive elements.[1].
These repetitive elements ...
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21
votes
Since every human has a different DNA (different combinations of C, G, A, T) what does it mean to have the genome done?
All humans have some differences in their DNA, but there's far more that is shared. On average the difference between humans is only about one thousandth of their full DNA, which means we're about 99....
- 311
19
votes
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What is the difference between a transcriptome and a genome?
In brief, the “genome” is the collection of all DNA present in the nucleus and the mitochondria of a somatic cell. The initial product of genome expression is the “transcriptome”, a ...
- 430
13
votes
What Ensembl genome version should I use for alignments? (e.g. toplevel.fa vs. primary_assembly.fa)
There's rarely a good reason to use a hard-masked genome (sometimes for blast, but that's it). For that reason, we use soft-masked genomes, which only have the benefit of showing roughly where repeats ...
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13
votes
Uppercase vs lowercase letters in reference genome
The use of lower/upper case letters and N/n letters in genomes sequences is not completely standardised and you should always ...
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12
votes
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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
10
votes
What is indel calling and what is its purpose?
There are two types of INDELs: short indels and long indels. Some put the threshold at 50bp; others choose 1000bp. Short and long indels are called differently.
<50bp short indels are called from ...
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10
votes
Why does the SARS-Cov2 genome has letter t
We sequence and therefore typically report assemblies as DNA sequences, even if they're actually RNA.
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9
votes
Accepted
What Ensembl genome version should I use for alignments? (e.g. toplevel.fa vs. primary_assembly.fa)
Generally, you should use the soft-masked or unmasked primary assembly. Cross-species whole-genome aligners, especially older ones, do need to know soft-masked regions; otherwise they can be ...
- 6,445
9
votes
Accepted
Aligning many long sequences
Whole genome aliment can be done using Progressive Mauve, LAST or Mummer. For bacteria I used Mauve since it has also very nice visualisation engine. A very new tool is Minimap2, a super fast mapper ...
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9
votes
Accepted
What is indel calling and what is its purpose?
Insertions and deletions (indels) are one type among many different types of genetic variation, such as single nucleotide variants (SNVs), copy number variants (CNVs), and structural variants (SVs). I'...
- 5,060
8
votes
Accepted
Way to get genomic sequences at given coordinates without downloading fasta files of whole chromosomes/genomes first?
using a http request.
if there is a DAS server, you can always use this protocol to download the xml -> fasta. see https://www.biostars.org/p/56/
...
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8
votes
Since every human has a different DNA (different combinations of C, G, A, T) what does it mean to have the genome done?
DNA is only the substance that makes up the genome.
DNA can come with many forms. For example, the fragments you get by PCR in the lab are DNA. A bacterium also has DNA molecules in it. You may also ...
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7
votes
How to use Python to count k-mers?
I wrote a command-line k-mer counter called kmer-counter that will output results in a form that your Python script can consume: https://github.com/alexpreynolds/...
- 3,125
7
votes
Why sequence the human genome at 30x coverage?
Solexa Inc. sequenced NA12878 chrX to ~30x in early 2007, which later became part of Bentley (2008). This, I believe, was the first time that 30x showed up. I don't recall they had a particular reason ...
- 6,445
7
votes
How do PCR duplicates arise and why is it important to remove them for NGS analysis?
PCR polymerases introduce errors. When an error arises in the first few cycles of amplifications, it will appear in a reasonably high fraction of DNA fragments in the library. After sequencing, you ...
- 6,445
7
votes
Accepted
dividing genome into non-overlapping windows using R
The question is a bit confusing to me, at it's core I understand you want non-overlapping windows across chromosomes.
One way to achieve is to use ...
- 600
7
votes
Accepted
Is this a low complexity region in our human genome?
Yes, that's a short low complexity region wedged between a SINE and an snRNA. More importantly, your alignments have MAPQ of 0 (that's why they're filled with white in IGV), which will happen if they ...
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7
votes
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How to calculate the memory usage of storing kmers in RAM
To follow up on Devon Ryan's answer, I thought it would be a little fun to write a Python script that demonstrates using a bit array to maintain a presence/absence table.
Note: I wrote a C++ port ...
- 3,125
7
votes
Accepted
How should the popular press compare similarity of genomes?
1–4% is from an evolution point of view. 99.7% is from a sequence point of view. Because they are measuring different things, they can be compatible with each other. The correct interpretation is: 1–4%...
- 6,445
6
votes
What is the difference between a transcriptome and a genome?
They are two very different things. Your genome is a large section of about 3 billion DNA nucleotide bases. It has no concept of exon and introns.
Transcriptome is a study of transcriptions. You have ...
- 2,739
6
votes
Why do BLASTn and prokka not seem to be searching the whole fasta file?
With a k-mer size of 28 it shouldn't be finding that many matches. And the prokka results are suspicious as well. Maybe you have multiple contigs (none larger than 100kb) in that file? What is the ...
- 61
6
votes
What is the standard way to work with a diploid reference genome? Complementary strands?
At the moment, the standard reference genomes (e.g. hg19, hg38) are haploid genomes. We know that the human genome is diploid. Naturally, the latter would be the respectively correct representation of ...
- 2,216
6
votes
Why sequence the human genome at 30x coverage?
30 times coverage is not unique to this problem, but number 30 has its empirical role in statistics:
In statistical analysis, the rule of three states that if a certain event did not occur in a ...
- 185
6
votes
Is this a low complexity region in our human genome?
Yes, this is a low complexity region.
Regions are considered low complexity (or having a simple sequence) when they contain an abundance of a single base, or an abundance of short tandem repeats. The ...
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6
votes
How to calculate the memory usage of storing kmers in RAM
I think that my calculations must be wrong. Otherwise, how could programs count kmers in RAM?
Hash table based k-mer counters only keep k-mers seen in data. For $16<k\le32$, you need 64-bit ...
- 6,445
6
votes
Accepted
Does the DNA or RNA of some ethnic groups map better than others' to the human reference genome sequence?
For simple variants like SNPs it would not really be a problem to use the current genome assembly for other ethnic groups. But for more complex variants this could be indeed problematic, however not ...
- 3,571
6
votes
Python API for working with ENSEMBL genomes
Now there's another option, the ensembl_rest module, a thin wrapper around the Ensembl REST API to simplify its usage and make it more pythonic. You can find the ...
- 61
6
votes
Accepted
full visualisation of draft genomes alignment
Found a solution, using D-Genies, worked great.
Some examples from their website:
Thanks to @user172818.
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