I have sequenced my phage genome from Qiagen. I'm using Galaxy CPT for genome assembly and annotation. My Question is that How can I confirm that I have assembled the contigs of my phage correctly?
Given that i have selected contig having the greater length and a coverage of 1000. Then I did blast on NCBI and e value of first selected sequences 35 is 0.0
Please help me out.
Hello Saleha,
There are different alternatives to evaluate the assembly using bioinformatics tools in general.
1- QUAST:
You can get a basic metrics of the assembly using this tool. In the results, you can see the how many contigs, N50 value, which is a quality metric in terms of contigs length in your assembly, the longest contig length and so on. However, N50 is like mean or median value so I would say N50 would not give you a powerful idea of the assembly.
2- BUSCOs (Benchmarking Universal Single-Copy Orthologs):
You can analyze your assembly in evolutionary core gene sets in different lineages. Basically, every genome must have some orthologous genes. You can see how many orthologous genes and how many of them are duplicated and fragmented in BUSCOs results. As you go down taxonomically, the number of genes in gene sets will of course increase.
3- REAPR (without reference):
It evaluates your genome assembly in terms of bases. You can see mis-assemblies, errors, error-free bases.
4- Reference genomes:
If you have somehow references or closely related genomes, you can mapped your genome against reference or closely related genome and have an idea how it looks.
As you can see, there is no only one way to assess your assembly if it is correct or not. More, those alternatives cannot say if your assembly is 100% correct or not. So, you can focus primarily on the compactness, in other words, less fragmented (you can use QUAST, and BUSCOs) then you can run REAPR.
Hope that helps. You can read following articles for more information.
Best,
References:
1- Gurevich A, Saveliev V, Vyahhi N, Tesler G (2013) QUAST: Quality assessment tool for genome assemblies.
2- Liao X, Li M, Zou Y, et al (2019) Current challenges and solutions of de novo assembly
3- Simão FA, Waterhouse RM, Ioannidis P, et al (2015) BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs
4- Hunt, M. et al. (2013) ‘REAPR: A universal tool for genome assembly evaluation’, Genome Biology.
5- Earl, D. et al. (2011) ‘Assemblathon 1: A competitive assessment of de novo short read assembly methods’
6- Bradnam, K. R. et al. (2013) ‘Assemblathon 2: Evaluating de novo methods of genome assembly in three vertebrate species’, GigaScience.
Uğur Çabuk Thankyou so much.
All of these are linux based. Can you suggest a window based tool?
Hello Saleha Masood
Unfortunately, I do not know If there is a windows-based tool. Also, most bioinformatics tools are linux-based. However, I would suggest that you can check Galaxy. Maybe you can do this type of analysis through one of Galaxy workflows.
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