Pangaea is designed to assemble short-reads with high specificity physical (linked-reads) or virtual barcodes (long-reads+short-reads). It includes (1) short-reads binning using variational autoencoder (2)multi-thresholding reassembly and (3) ensemble assembly.
We have built a docker image for users to directly run Pangaea (without automaticly select cluster numbers).
docker pull jmelody/pangaea:std
git clone https://github.com/ericcombiolab/Pangaea.git
nohup docker run -v $PWD/Pangaea/example/:/example -u $(id -u):$(id -g) jmelody/pangaea:std /bin/bash /app/run_test.sh -1 /example/reads1.fq.gz -2 /example/reads2.fq.gz -sp /example/contigs.fa -lc /example/flye-input-contigs.fa -at /example/athena.asm.fa -o /example/pangaea -c 5 &Please remember set -u $(id -u):$(id -g) to avoid using root user. Otherwise the Pangaea/example directory will also be root permission.
If you got error when running Docker. Please: 1. check the log file example/pangaea/log. You can also open a issue and share the log file with us.
Or, 2. you can run/follow the script build.sh which will create a conda envrionment and build the necessary packages step by step.
An all-in-one installation script. This may take about 20 minutes ~ 1 hours.
Note! This script will download MetaPhlan4's latest database into this directory $PWD/metaplan4_DB, which requires about 20G storage and the process may take long. If you would like to specify a dedicated path for this, please run ./build.sh -d [path] instead.
Suggestion: MetaPhlan4 is used for clustering number choosing, if you tend to specify a clustering number by your own, you can just skip this installation step and run pangaea with -c [number] option. According to our experiments, we suggest using a number around 35 as the default c for complexed metagenomic data( in which their species number is above 200 and shannon diversity is around 3.5 ). For mock or simulation data, we suggest using a smaller or equal number to the real species number if the species number is lower than 35.
git clone https://github.com/ericcombiolab/Pangaea.git
cd Pangaea
./build.sh
# set up metaphlan4
./build_db.sh (this will download the database in current directory named ./metaphlan4_DB)
# or
./build_db.sh -d [your prefered directory(please make sure the space is larger than 25G)]Pangaea depends on numpy >= 1.23.5, pandas >= 1.5.3, sklearn >= 1.2.2, snakemake, pysam >= 0.20.0, torch 1.10.0, rph_kmeans, pigz 2.4, bwa >= 0.7.17, samtools 1.9, seqtk, megahit v1.2.9, spades(>=v3.15.3), flye 2.8-b1674, quickmerge, Jellyfish 2.3.0and jgi_summarize_bam_contig_depths.
Examples and experiments run on a Linux server with the following specifications:
Hardware:
Software:
./build.sh ）conda env create -f environment.yaml
conda activate pangaea
# Optional: Install Athena (based on python2), run
conda env creat -f athena_enviroment.ymal
conda activate athenaNote that if you want to run Athena/Pangaea, you need to change conda enviroments accrodingly.
./build.sh ）cd Pangaea/cpptools && make && cd -Run metaspades to obtain error-corrected reads and seed contigs for Athena and Pangaea:
metaspades.py --12 /path/to/reads -o /path/to/metaspades/outThe reads1 (input to Pangaea -1), reads2 (input to Pangaea -2) are in /path/to/metaspades/out/corrected/ and spades contigs (input to Pangaea -sp) are at /path/to/metaspades/out/contigs.fasta.
Run athena to obtain local assembly contigs and athena contigs. The local assembly contigs (input to Pangaea -lc) and athena contigs (input to Pangaea -at) are /path/to/athena/out/results/olc/flye-input-contigs.fa and /path/to/athena/out/results/olc/athena.asm.fa.
usage: pangaea.py [-h] [-1 READS1] [-2 READS2] [-i INTERLEAVED_READS] -o
OUTPUT [-l MIN_LENGTH] [-k KMER] [-tnf_k TNF_KMER]
[-s WINDOW_SIZE] [-v VECTOR_SIZE] [-r LR] [-w WEIGHT_DECAY]
[-e EPOCHS] [-b BATCH_SIZE] [-d DROPOUT] [-p PATIENCE]
[-wa WEIGHT_ALPHA] [-wk WEIGHT_KL] [-ld LATENT_DIM] -c
CLUSTERS [-t THREADS] [-g USE_CUDA] [-n NUM_GPUS]
[-sp SPADES] [-lc LOCAL_ASSEMBLY] [-at ATHENA]
[-lt LOW_ABD_CUT] [-la LOW_ASSEMBLER] [-md MODEL]
[-ls LOSS_TYPE] [-st STEPS]
optional arguments:
-h, --help show this help message and exit
-1 READS1, --reads1 READS1
path to reads1 file (linked-reads)
-2 READS2, --reads2 READS2
path to reads2 file (linked-reads)
-i INTERLEAVED_READS, --interleaved_reads INTERLEAVED_READS
path to reads file (long-reads)
-o OUTPUT, --output OUTPUT
output directory
-l MIN_LENGTH, --min_length MIN_LENGTH
min barcode length (default 2000)
-k KMER, --kmer KMER kmer for abundance (default 15)
-tnf_k TNF_KMER, --tnf_kmer TNF_KMER
kmer for TNF (default 4, long reads should use 3)
-s WINDOW_SIZE, --window_size WINDOW_SIZE
window size for abundance (default 10)
-v VECTOR_SIZE, --vector_size VECTOR_SIZE
vector size for abundance (default 400)
-r LR, --lr LR learning rate (default 0.005)
-w WEIGHT_DECAY, --weight_decay WEIGHT_DECAY
weight decay (default 0.0001)
-e EPOCHS, --epochs EPOCHS
number of epochs (default 100)
-b BATCH_SIZE, --batch_size BATCH_SIZE
batch size (default 2048)
-d DROPOUT, --dropout DROPOUT
dropout (default 0.2)
-p PATIENCE, --patience PATIENCE
early stop patience (default 20)
-wa WEIGHT_ALPHA, --weight_alpha WEIGHT_ALPHA
training weight for abundance and tnf (default 0.1)
-wk WEIGHT_KL, --weight_kl WEIGHT_KL
training weight for KL (default 0.015)
-ld LATENT_DIM, --latent_dim LATENT_DIM
latent dimension (default 32)
-c CLUSTERS, --clusters CLUSTERS
number of clusters
-t THREADS, --threads THREADS
number of threads (default 100)
-g USE_CUDA, --use_cuda USE_CUDA
use cuda (default False)
-n NUM_GPUS, --num_gpus NUM_GPUS
use gpu in parallel (if use cuda)
-sp SPADES, --spades SPADES
path to original contigs
-lc LOCAL_ASSEMBLY, --local_assembly LOCAL_ASSEMBLY
path to local assembly contigs
-at ATHENA, --athena ATHENA
path to athena contigs
-lt LOW_ABD_CUT, --low_abd_cut LOW_ABD_CUT
coverage for low abundance contigs
-la LOW_ASSEMBLER, --low_assembler LOW_ASSEMBLER
local assembly method (spades or megahit)
-md MODEL, --model MODEL
model ( vae)
-ls LOSS_TYPE, --loss_type LOSS_TYPE
reconstruction loss type (default ce)
-st STEPS, --steps STEPS
steps to run (default 1:feature extraction, 2:vae trainning, 3:clutsering, 4:sub-assembly and final
assembly)conda activate pangaea
cd example
nohup python ../pangaea.py -1 reads1.fq.gz -2 reads2.fq.gz -sp contigs.fa -lc flye-input-contigs.fa -at athena.asm.fa -c 5 -o pangaea > pangaea.log 2>&1 &The generated final assembly will be at pangaea/final.asm.fa.
This may take about 1~2 hours.
cd example/hybrid
nohup ./hybrid_wrapper.sh atcc_longreads_small.fastq.gz atcc_short_R1.fastq.gz atcc_short_R2.fastq.gz 60 operams > hybrid.log 2>&1 &The generated final assembly will be at pangaea_out/final.asm.fa.
This may take about 1~2 hours.
# type: operams, hybridspades
./final_merge.sh <type>The new generated final assembly will be at pangaea_out/4.assembly/quickmerge_<type>/merged_out.fasta.
This may take about 10~30 minutes.