Sequencing the genome of the freshwater angelfish

Methods

Summary

Broadly the steps of this work are as following: 

1. Extract DNA 

2. Prepare DNA ligation library 

3. Load into Nanopore Device 

4. Sequence and Collect Reads. 

5. Check for contamination of Reads 

6. Assemble Reads. 

7. Calculate BUSCO score 

8. Remove foreign contigs 

9. Deposit to NCBI. 

But do note that I have already successfully completed all of the above and I am raising funds to improve the assembly. 

Challenges

The only challenge I have had so far was extracting angelfish genomic DNA with the NEB high molecular weight  DNA extraction kit. However, after switching over to the NEB genomic purification kit, I was successfully able to extract 6µg of DNA at an average concentration of 30ng/µL from the angelfish tissue which is enough for a total of 6 sequencing runs. 

My personal computer also did not have enough memory to perform a de novo assembly of the collected reads. However, I was successfully able to assemble the partial genome after switching over to the Texas Advanced Computing Center's Stampede supercomputer. Assembly however also often takes around 3-4 hours to complete.

Pre Analysis Plan

Analysis of assembly will be performed by calculating a BUSCO score. Essentially the BUSCO tool searches the flye assembly to identify highly conserved fish genes. The more genes that are found, the more likely that the genome was properly and completely assembled. I currently have a BUSCO score of 65% on my current genome assembly for this fish.