INTRO

As part of project-lake-trout (GitHub repo), Steven asked that I assemble the genome of a lean 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-lean.Rmd. The run took ~7 days.

PRIMARY OUTPUTS

FastA (3.9GB): https://gannet.fish.washington.edu/gitrepos/project-lake-trout/output/13.1-genome-assembly-lean/snam-lean-pb-flye-assembly.fasta
MD5: https://gannet.fish.washington.edu/gitrepos/project-lake-trout/output/13.1-genome-assembly-lean/snam-lean-pb-flye-assembly.fasta.md5
FastA index: https://gannet.fish.washington.edu/gitrepos/project-lake-trout/output/13.1-genome-assembly-lean/snam-lean-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.

13.1-genome-assembly-lean

Sam White 2026-03-26

1 BACKGROUND

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

2 SETUP

2.1 Libraries

library(knitr)
library(tidyverse)

## ── Attaching core tidyverse packages ──────────────────────── tidyverse 2.0.0 ──
## ✔ dplyr     1.1.4     ✔ readr     2.1.5
## ✔ forcats   1.0.0     ✔ stringr   1.5.1
## ✔ ggplot2   3.5.2     ✔ tibble    3.2.1
## ✔ lubridate 1.9.4     ✔ tidyr     1.3.1
## ✔ purrr     1.0.4     
## ── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
## ✖ dplyr::filter() masks stats::filter()
## ✖ dplyr::lag()    masks stats::lag()
## ℹ Use the conflicted package (<http://conflicted.r-lib.org/>) to force all conflicts to become errors

library(reticulate)

knitr::opts_chunk$set(
  echo = TRUE,         # Display code chunks
    eval = FALSE,        # Evaluate code chunks
    results = "hold",   # Hold outputs and show them after the full code chunk
  warning = FALSE,     # Hide warnings
        collapse = FALSE,    # Keep code and output in separate blocks
        warning = FALSE,     # Hide warnings
        message = FALSE,     # Hide messages
        comment = "##"      # Prefix output lines with '##' so output is visually distinct
)

2.2 Set R variables

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

#OUTPUT FILE(S)
prefix <- "snam-lean-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 <- "32"


# 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

# 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-lean-assembly.log 2>&1

3 OUTPUTS

3.1 List files

ls -ltrh "${output_dir}"

## total 8.2G
## -rw-r--r-- 1 samwhite all  326 Mar 26 11:02 input_fastqs.txt
## drwxr-xr-x 2 samwhite all  512 Mar 30 07:44 00-assembly
## drwxr-xr-x 2 samwhite all 8.0K Mar 30 12:12 10-consensus
## drwxr-xr-x 2 samwhite all  512 Mar 31 08:22 20-repeat
## drwxr-xr-x 2 samwhite all  512 Mar 31 08:33 30-contigger
## drwxr-xr-x 2 samwhite all 8.0K Mar 31 17:25 40-polishing
## -rw-r--r-- 1 samwhite all   92 Mar 31 17:25 params.json
## -rw-r--r-- 1 samwhite all  53M Mar 31 17:25 assembly_graph.gv
## -rw-r--r-- 1 samwhite all 4.0G Mar 31 17:25 assembly_graph.gfa
## -rw-r--r-- 1 samwhite all 3.9G Mar 31 17:25 assembly.fasta
## -rw-r--r-- 1 samwhite all 4.8M Mar 31 17:25 assembly_info.txt
## -rw-r--r-- 1 samwhite all 3.2K Mar 31 17:25 pb-lean-assembly.log
## -rw-r--r-- 1 samwhite all 338M Mar 31 17:25 flye.log
## -rw-r--r-- 1 samwhite all 3.9M Apr  2 10:05 assembly.fasta.fai

3.2 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

3.3 Create FastA Index

cd "${output_dir}"

samtools faidx ${prefix}-assembly.fasta

3.4 Create checksums

cd "${output_dir}"

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

## ac7c2af6c32834f86954cfb9893c4c50  snam-lean-pb-flye-assembly.fasta
## 242ea029692207d1c7df0b7e842fd58f  snam-lean-pb-flye-assembly.fasta.fai
## 35f1973b873fd7498ebeeecdb7eecd2e  snam-lean-pb-flye-assembly_graph.gfa
## d4f6a3a41846f6285d0290c7a41ed1f7  snam-lean-pb-flye-assembly_graph.gv
## e1224b9a628253fd8cee1d39517343ef  snam-lean-pb-flye-assembly_info.txt

3.5 Assembly stats

cd "${output_dir}"

tail flye.log

## [2026-03-31 17:25:45] root: INFO: Assembly statistics:
## 
##  Total length:   4046369763
##  Fragments:  112562
##  Fragments N50:  74514
##  Largest frg:    2170611
##  Scaffolds:  0
##  Mean coverage:  15
## 
## [2026-03-31 17:25:45] root: INFO: Final assembly: /mmfs1/gscratch/scrubbed/samwhite/gitrepos/RobertsLab/project-lake-trout/output/13.1-genome-assembly-lean/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.