INTRO

As part of project-lake-trout (GitHub repo), Steven asked that I assemble the genome of a siscowet ecotype of lake trout (GitHub Issue), Salvelinus namaycush, using PacBio HiFi reads. I opted to use Flye for the assembly, as we’d previously used it for Chionoecetes bairdi (Tanner crab) (Wikipedia), and it performed well. The assembly process was straightforward and quick.

I performed the assembly on Hyak (Klone), UW’s high-performance computing cluster. I used our Apptainer (Singularity) container to run the job (srlab-R4.4-bioinformatics-container-fca8ae6.sif), which includes Flye version 2.9.6.

Below is the rendered markdown from 13.1-genome-assembly-siscowet.Rmd. The run took ~7 days.

PRIMARY OUTPUTS

FastA (4.1GB): https://gannet.fish.washington.edu/gitrepos/project-lake-trout/output/13.1-genome-assembly-siscowet/snam-siscowet-pb-flye-assembly.fasta
MD5: https://gannet.fish.washington.edu/gitrepos/project-lake-trout/output/13.1-genome-assembly-siscowet/snam-siscowet-pb-flye-assembly.fasta.md5
FastA index: https://gannet.fish.washington.edu/gitrepos/project-lake-trout/output/13.1-genome-assembly-siscowet/snam-siscowet-pb-flye-assembly.fasta.fai

Once I’ve also assembled this genome using hifiasm, I’ll compare the two assemblies using QUAST, which will provide a more comprehensive set of assembly statistics and metrics.

1 BACKGROUND

Use Flye(GitHub) (Kolmogorov et al. 2019) to assemble PacBio reads for S.namaycush siscowet ecotype.

2 SETUP

2.1 Libraries

2.2 Set R variables

# OUTPUT DIRECTORY
data_dir <- "../data/pacbio-siscowet"
output_dir <- "../output/13.1-genome-assembly-siscowet"

#OUTPUT FILE(S)
prefix <- "snam-siscowet-pb-flye"



# CONDA
conda_env_name <- c("/srlab/programs/miniforge3-24.7.1-0/envs/flye-2.9.6-env")
conda_path <- c("/srlab/programs/miniforge3-24.7.1-0/bin/conda")

# SETTINGS
genome_size <- "2.3g"
threads <- "100"


# Export these as environment variables for bash chunks.
Sys.setenv(
  data_dir = data_dir,
  genome_size = genome_size,
  output_dir = output_dir,
  prefix = prefix,
  threads = threads
)

2.3 Load Flye conda environment

If this is successful, the first line of output should show that the Python being used is the one in your Flye conda environment path.

E.g.

python: /srlab/programs/miniforge3-24.7.1-0/envs/flye-2.9.6-env/bin/python

use_condaenv(condaenv = conda_env_name, conda = conda_path)
py_config()

## python:         /srlab/programs/miniforge3-24.7.1-0/envs/flye-2.9.6-env/bin/python
## libpython:      /srlab/programs/miniforge3-24.7.1-0/envs/flye-2.9.6-env/lib/libpython3.12.so
## pythonhome:     /srlab/programs/miniforge3-24.7.1-0/envs/flye-2.9.6-env:/srlab/programs/miniforge3-24.7.1-0/envs/flye-2.9.6-env
## version:        3.12.13 | packaged by conda-forge | (main, Mar  5 2026, 16:50:00) [GCC 14.3.0]
## numpy:           [NOT FOUND]
## 
## NOTE: Python version was forced by use_python() function

3 FLYE

# Make output directory, if it doesn't exist
mkdir --parents "${output_dir}"

# Create an array of .fastq.gz files
fastqs=(${data_dir}/*.fastq.gz)

# Print the result (optional, for verification)
## newline-delimited format
echo "List of FastQs used for assembly:"

printf "%s\n" "${fastqs[@]}" \
| tee "${output_dir}/input_fastqs.txt"

# Run Flye assembly
flye \
--pacbio-hifi "${fastqs[@]}" \
--genome-size "${genome_size}" \
--out-dir "${output_dir}" \
--threads "${threads}" \
> "${output_dir}"/pb-siscowet-assembly.log 2>&1

4 OUTPUTS

4.1 List files

ls -ltrh "${output_dir}"

## total 8.9G
## -rw-r--r-- 1 samwhite all  342 Apr  6 08:08 input_fastqs.txt
## drwxr-xr-x 2 samwhite all  512 Apr  8 11:51 00-assembly
## drwxr-xr-x 2 samwhite all 8.0K Apr  8 15:08 10-consensus
## drwxr-xr-x 2 samwhite all  512 Apr  9 02:23 20-repeat
## drwxr-xr-x 2 samwhite all  512 Apr  9 02:40 30-contigger
## drwxr-xr-x 2 samwhite all 8.0K Apr  9 10:29 40-polishing
## -rw-r--r-- 1 samwhite all   92 Apr  9 10:29 params.json
## -rw-r--r-- 1 samwhite all  55M Apr  9 10:29 assembly_graph.gv
## -rw-r--r-- 1 samwhite all 4.3G Apr  9 10:30 assembly_graph.gfa
## -rw-r--r-- 1 samwhite all 4.2G Apr  9 10:30 assembly.fasta
## -rw-r--r-- 1 samwhite all 4.8M Apr  9 10:30 assembly_info.txt
## -rw-r--r-- 1 samwhite all 3.2K Apr  9 10:30 pb-siscowet-assembly.log
## -rw-r--r-- 1 samwhite all 482M Apr  9 10:30 flye.log

4.2 Rename files

Files have generic “assembly_*” naming.

Renaming to reflect specific species-ecotype-data-assembly_method.

cd "${output_dir}"

for file in assembly*
do
  mv "${file}" ${prefix}-${file}
done

4.3 Create FastA Index

cd "${output_dir}"

samtools faidx ${prefix}-assembly.fasta

4.4 Create checksums

cd "${output_dir}"

for file in ${prefix}*
do
  md5sum "${file}" \
  | tee "${file}".md5
done

## 30139d7d8d7fdad878543fc5a5f3f0a8  snam-siscowet-pb-flye-assembly.fasta
## adf9dfc0996f12c28a41089e148e693a  snam-siscowet-pb-flye-assembly.fasta.fai
## 62f9467fdd049e7dafaf7c63871c4184  snam-siscowet-pb-flye-assembly_graph.gfa
## 949d7663ab4d54500e00856fd80deed9  snam-siscowet-pb-flye-assembly_graph.gv
## 1f336e131d47f582e15bdd0acc66414f  snam-siscowet-pb-flye-assembly_info.txt

4.5 Assembly stats

cd "${output_dir}"

tail flye.log

## [2026-04-09 10:30:36] root: INFO: Assembly statistics:
## 
##  Total length:   4336778941
##  Fragments:  110158
##  Fragments N50:  78564
##  Largest frg:    2753427
##  Scaffolds:  0
##  Mean coverage:  21
## 
## [2026-04-09 10:30:36] root: INFO: Final assembly: /mmfs1/gscratch/scrubbed/samwhite/gitrepos/RobertsLab/project-lake-trout/output/13.1-genome-assembly-siscowet/assembly.fasta

Kolmogorov, Mikhail, Jeffrey Yuan, Yu Lin, and Pavel A. Pevzner. 2019. “Assembly of Long, Error-Prone Reads Using Repeat Graphs.” Nature Biotechnology 37 (5): 540–46. https://doi.org/10.1038/s41587-019-0072-8.