Choosing Optimal Assembly from Quast Data

[richelleredekop]

Hello Everyone,

I am very new at next generation sequencing and have a question about choosing which assembly is the best to use going forward. My samples are isolates of Helicobacter pylori that I have sent for whole genome sequencing. I have paired end Illumina reads and used Trimmomatic to process them and FastQC to make sure everything appeared acceptable. I then tried deNovo assembly of the forward and reverse paired reads using Velvet, Abyss and SPAdes. I then took the contig files produced from these 3 assembly methods and ran them through Quast to evaluate which assembly worked the best. I have attached links to the alignment produced and the summary file.

Alignment:

https://drive.google.com/file/d/0B1G2M5ad3_x4Vm0tbzBhMGJSVVk/view?usp=sharing

Summary File

https://drive.google.com/file/d/0B1G2M5ad3_x4amtFc3RiNkxRMVE/view?usp=sharing

Abyss and Spades had similar output, with SPAde perhaps being marginally better based on # contigs, largest contig, and N50. Velvet was quite different from Abyss and SPAde and had much fewer misassemblies (6 vs 35 for AByss and 31 for SPAdes). I am not sure what would account for this large difference.

If anyone could point me in the right direction as to which assembly is the best to use and/or how to improve my assemblies I would really appreciate it. Like I said I am super to to NGS and have limited computing skills so this has been a huge learning experience for me (but a fun one!).

Thanks in advance!

[Brian Bushnell]

Velvet has a much lower misassembly rate because it is less aggressive and produced a much more fragmented assembly (N50=3862 compared to 141550 for Spades).

I tend to look at the number of predicted long genes (>3000 and >1500bp) as an indicator of assembly quality. Here, Abyss and Spades are similar and Velvet is very much inferior.

It's kind of a toss-up whether Abyss or Spades assembly is better. Spades is more continuous with slightly fewer misassemblies, so I'd probably favor that.

[richelleredekop]

Thank you for your reply, I appreciate you taking the time to help me out. I was leaning towards Spades but Velvet being so different was throwing me off. I will go with Spades, which is what my gut was telling me but as I am so new I wanted to make sure I was making the right call. Thank you!