From looking at the genome browser on NCBI (https://www.ncbi.nlm.nih.gov/genome/browse#!/overview/ ) among other reading I have found it difficult to distinguish between the different "grades" of genome completeness.
Complete
Chromosome
Scaffold
Contig
etc.
For example when is it scaffolded enough to be a chromosome.
For reference I am coming from a bacterial angle so multiple chromosomes don't effect me much, though permanent plasmids might. I've done a bit of genome assembly so am looking to both understand other peoples data and understand at what grade mine would fit in.
1) I have found it difficult to draw sharp delineations at when a genome might fit the different criteria. any useful rules of thumb or guides?
2) is there a point at which you might consider a genome trustworthy for example for whole genome alignment, or confirming the existence or absence of genes.
3) Some genomes are referred to as "permanent draughts" in literature which I assumes means they will never be complete but I don't quite understand why.
Hi Benedict Claxton Stevens , I don't have much experience with bacterial genome assembly but have worked on a eukaryotic reference assembly (Swiss Alpine whitefishArticle A de novo chromosome‐level genome assembly of Coregonus sp. ...
). I think you raise a lot of interesting points and after going through the process of assembling a reference genome I can try to maybe give my take on your questions - sorry for the essay...1) For me the 'easiest' to justify would be contig and scaffold. In my mind contigs result from a first round of assembly i.e. sequenced reads are processed to find overlaps and these are merged into contigs. Then, in my mind, scaffolds are contigs which have been joined together in some way with additional evidence, this could be linkage evidence by using a linkage map and finding markers which sit together on linkage groups or through something like Hi-C which rather uses physical linkage. Chromosome-level assemblies become a bit of a semantic argument i'd say and I presume most people go through some process of having to justify why they think their assembly is/isn't chromosome scale. I think saying something is chromosome-scale depends on knowing that your linkage map linkage groups/Hi-C data has resolved the number of chromosomes you observe from karyotype data for example, as well as knowing that your assembly length is close to the known length of the genome (e.g. calculated using an estimate produced from a k-mer approach - like with GenomeScope or knowing the mass). The boldest of the terms, 'complete', in my mind is not particularly helpful biologically since I'm certain that most assemblies do not contain all nucleotides and cannot contain all structural variants. But I presume most people who use this term have carried out substantially deeper sequencing and have tried numerous iterative approaches to get as close to the 'true' genome as possible. These references may also be qualified as 'complete' based off of known gene content, e.g. if they have close to 100% BUSCO scores.
2) With regards to your second question I'd say that seeing how much raw sequencing data maps to an assembly is a good estimate of its accuracy/usefulness - although this approach definitely works best if you have a few options and can pick the best one. I think the same is true of gene completeness where you could look at BUSCO scores for each and then see which are closest to complete for genes which should definitely be there.
3) I think permanent drafts could just mean that they are not going to sequence any more from the same individual/clone e.g. they won't add more PacBio/Hi-C data now to marginally improve the assembly, but that the quality is good enough and widely used enough to be used as-is.
I hope this helps!
Rishi
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Assembly level - the highest level of assembly for any object in the assembly:
- Complete genome - all chromosomes are gapless and have no runs of 10 or more ambiguous bases (Ns), there are no unplaced or unlocalized scaffolds, and all the expected chromosomes are present (i.e. the assembly is not noted as having partial genome representation). Plasmids and organelles may or may not be included in the assembly but if present then the sequences are gapless.
- Chromosome - there is sequence for one or more chromosomes. This could be a completely sequenced chromosome without gaps or a chromosome containing scaffolds or contigs with gaps between them. There may also be unplaced or unlocalized scaffolds.
- Scaffold - some sequence contigs have been connected across gaps to create scaffolds, but the scaffolds are all unplaced or unlocalized
- Contig - nothing is assembled beyond the level of sequence contigs
from https://www.ncbi.nlm.nih.gov/assembly/help/
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