Introduction

NECAT is an error correction and de-novo assembly tool for Nanopore long noisy reads.

If you are interested in calling Structural Variants from Nanopore reads, you are welcome to have a try our necatsv.

Installation

We have sucessfully tested NECAT on

• Ubuntu 16.04 (GCC 5.4.0, Perl v5.22.1)
• CentOS 7.3.1611 (GCC 4.8.5, Perl v5.26.2)

If you meet problems in running NECAT like

Syntax error at NECAT/Linuax-amd64/bin/Plgd/Project.pm line 46, near "${cfg{"

Please update your perl to a newer version (such as v5.26).

There are two ways to install NECAT.

Install from executable binaries

$ wget https://github.com/xiaochuanle/NECAT/releases/download/v0.0.1_update20200803/necat_20200803_Linux-amd64.tar.gz
$ tar xzvf necat_20200803_Linux-amd64.tar.gz
$ cd NECAT/Linux-amd64/bin
$ export PATH=$PATH:$(pwd)

Build from source codes

$ git clone https://github.com/xiaochuanle/NECAT.git
$ cd NECAT/src/
$ make
$ cd ../Linux-amd64/bin
$ export PATH=$PATH:$(pwd)

After installation, all the executable files can be found in NECAT/Linux-amd64/bin. The command line

export PATH=$PATH:$(pwd)

above is used for adding NECAT/Linux-amd64/bin to the system PATH.

Quick Start

Before running NECAT please do not forget to add NECAT/Linux-amd64/bin to the system PATH.

Step 1: Create a config file

Create a config file template using the following command:

$ necat.pl config ecoli_config.txt

The template looks like

PROJECT=
ONT_READ_LIST=
GENOME_SIZE=
THREADS=4
MIN_READ_LENGTH=3000
PREP_OUTPUT_COVERAGE=40
OVLP_FAST_OPTIONS=-n 500 -z 20 -b 2000 -e 0.5 -j 0 -u 1 -a 1000
OVLP_SENSITIVE_OPTIONS=-n 500 -z 10 -e 0.5 -j 0 -u 1 -a 1000
CNS_FAST_OPTIONS=-a 2000 -x 4 -y 12 -l 1000 -e 0.5 -p 0.8 -u 0
CNS_SENSITIVE_OPTIONS=-a 2000 -x 4 -y 12 -l 1000 -e 0.5 -p 0.8 -u 0
TRIM_OVLP_OPTIONS=-n 100 -z 10 -b 2000 -e 0.5 -j 1 -u 1 -a 400
ASM_OVLP_OPTIONS=-n 100 -z 10 -b 2000 -e 0.5 -j 1 -u 0 -a 400
NUM_ITER=2
CNS_OUTPUT_COVERAGE=30
CLEANUP=1
USE_GRID=false
GRID_NODE=0
GRID_OPTIONS=
SMALL_MEMORY=0
FSA_OL_FILTER_OPTIONS=
FSA_ASSEMBLE_OPTIONS=
FSA_CTG_BRIDGE_OPTIONS=
POLISH_CONTIGS=true

Filling and modifying the relative information, we have

PROJECT=ecoli
ONT_READ_LIST=read_list.txt
GENOME_SIZE=4600000
THREADS=20
MIN_READ_LENGTH=3000
  ......

read_list.txt in the second line above contains the full paths of all read files. It looks like

$ cat read_list.txt
/share/home/chuanlex/xiaochuanle/data/testdata/tomato/20161027_Spenn_001_001_all.fastq
/share/home/chuanlex/xiaochuanle/data/testdata/tomato/20161101_Spenn_002_002_all.fastq
/share/home/chuanlex/xiaochuanle/data/testdata/tomato/20161103_Spenn_003_003_all.fastq
/share/home/chuanlex/xiaochuanle/data/testdata/tomato/20161108_Spenn_004_004_all.fastq
/share/home/chuanlex/xiaochuanle/data/testdata/tomato/20161108_Spenn_004_005_all.fastq

Please note that files in read_list.txt need not be the same format. Each file can independently be either FASTA or FASTQ, and can further be compressed in GNU Zip (gzip) format.

Step 2: Correct raw reads

Correct the raw noisy reads using the following command:

$ necat.pl correct ecoli_config.txt

The pipeline only corrects longest 40X (PREP_OUTPUT_COVERAGE) raw reads. The corrected reads are in the files ./ecoli/1-consensus/cns_iter${NUM_ITER}/cns.fasta.
The longest 30X (CNS_OUTPUT_COVERAGE) corrected reads are extracted for assembly, which are in the file ./ecoli/1-consensus/cns_final.fasta

Step 3: Assemble contigs

After correcting the raw reads, we assemble the contigs using the following command. If the correcting-step is not done, the command automatically runs the correcting-step first.

$ necat.pl assemble ecoli_config.txt

The assembled contigs are in the file ./ecoli/4-fsa/contigs.fasta.

Step 4: Bridge contigs

After assembling the contigs, we run the bridging-step using the following command. The command checks and runs the preceding steps first.

$ necat.pl bridge ecoli_config.txt

The bridged contigs are in the file ./ecoli/6-bridge_contigs/bridged_contigs.fasta.

If POLISH_CONTIGS is set, the pipeline uses the corrected reads to polish the bridged contigs. The polished contigs are in the file ./ecoli/6-bridge_contigs/polished_contigs.fasta

Running with multiple computation nodes

On PBS and SGE systems, users may plan to run NECAT with multiple computation nodes. This is done by setting the config file (Step 1 of Quick Start) like

USE_GRID=true
GRID_NODE=4

In the above example, 4 computation nodes will be used and each computation node will run with THREADS CPU threads.

Citation

Chen Y, Nie F, Xie S Q, et al. Efficient assembly of nanopore reads via highly accurate and intact error correction[J]. Nature Communications, 2021, 12(1): 1-10.

Contact

• Chuan-Le Xiao, [email protected]