Contents

• Updates in 2020.02.28

• Introduction

• Installation

• Quick Start

• Input Format

• Program Descriptions

• Citation

• Contact

• Update Information

Updates of 2020.02.28

• Removed hard limit on maximum sequence length. The hard limit always causes segmentation fault when performing ultra long sequence alignments.

• Merge mecat2pw and mecat2ref into one single mapping tool mecat2map. mecat2map uses much less memory compared to mecat2pw.

• The candidate partition process now supports multiple CPU threads.

• Support multiple input forms (see Input Format).

Introduction

MECAT2 is an improved version of MECAT. It is an ultra-fast and accurate Mapping, Error Correction and de novo Assembly Tools for single molecula sequencing (SMRT) reads.

MECAT2 consists of three modules:

• mecat2map, a fast and accurate alignment tool for SMRT reads

• mecat2cns, correct noisy reads based on their pairwise overlaps

• fsa, a string graph based assembly tool.

MECAT2 is written in C, C++, and perl. It is open source and distributed under the GPLv3 license.

Please note that MECAT2 no longer supports Nanopore raw reads. We have developed a Mapping, Error Correction and de novo Assembly Pipeline specifically for Nanopore Raw Reads NECAT. follow this link to NECAT.

Installation

We have tested MECAT2 on CentOS release 7.3 and on Ubuntu 18.04.

• Step 1: Figure out where to install MECAT2. We will install MECAT2 and two other auxiliary tools HDF5 and dextract. We first identify the directory in which we want to install them. As an example, I will install them in the directory /home/chenying/smrt_asm. So I first create this directory using the mkdir command and go to that directory: (The dollar sign $ that preceeds the input is the promt printed by the shell.)

$ mkdir -p /home/chenying/smrt_asm
$ cd /home/chenying/smrt_asm
$ pwd
/home/chenying/smrt_asm

For easy reference, we asign /home/chenying/smrt_asm to an environment variable MECAT_PATH:

$ export MECAT_PATH=/home/chenying/smrt_asm
$ echo ${MECAT_PATH}
/home/chenying/smrt_asm

• Step 2: Install MECAT2:

$ git clone https://github.com/xiaochuanle/MECAT2.git
$ cd MECAT2
$ make 
$ cd ..

After installation, all the executables are found in ${MECAT_PATH}/MECAT/Linux-amd64/bin. The folder name Linux-amd64 will vary in operating systems.

• Step 3: Add relative pathes

$ export PATH=${MECAT_PATH}/MECAT/Linux-amd64/bin:$PATH

Quick Start

Before running MECAT2, don't forget to add binary paths to PATH (Step 3 of Installation).

Here we take assemblying the genome of Ecoli as an example, to go through each step in order. Details of each step are given in the next section.

• Step 1: Download dataset. We download the raw reads ecoli_filtered.fastq.gz into directory ${MECAT_PATH}/ecoli

$ mkdir -p ${MECAT_PATH}/ecoli
$ cd ${MECAT_PATH}/ecoli
$ wget http://gembox.cbcb.umd.edu/mhap/raw/ecoli_filtered.fastq.gz

After that, we get raw read file ${MECAT_PATH}/ecoli/ecoli_filtered.fastq.gz:

$ ls
ecoli_filtered.fastq.gz

• Step 2: Prepare config file We create a config file template using the following command:

$ mecat.pl config ecoli_config_file.txt

This command creates a config file ecoli_config_file.txt, which looks like

PROJECT=
RAWREADS=
GENOME_SIZE=
THREADS=4
MIN_READ_LENGTH=2000
CNS_OVLP_OPTIONS="-kmer_size 13"
CNS_PCAN_OPTIONS="-p 100000 -k 100"
CNS_OPTIONS=""
CNS_OUTPUT_COVERAGE=30
TRIM_OVLP_OPTIONS="-skip_overhang"
TRIM_PM4_OPTIONS="-p 100000 -k 100"
TRIM_LCR_OPTIONS=""
TRIM_SR_OPTIONS=""
ASM_OVLP_OPTIONS=""
FSA_OL_FILTER_OPTIONS="--max_overhang=-1 --min_identity=-1"
FSA_ASSEMBLE_OPTIONS=""
CLEANUP=0

After filling the relative information, we have

PROJECT=ecoli
RAWREADS=/home/chenying/smrt_asm/ecoli/ecoli_filtered.fastq
GENOME_SIZE=4800000
THREADS=4
MIN_READ_LENGTH=2000
CNS_OVLP_OPTIONS="-kmer_size 13"
CNS_PCAN_OPTIONS="-p 100000 -k 100"
CNS_OPTIONS=""
CNS_OUTPUT_COVERAGE=30
TRIM_OVLP_OPTIONS="-skip_overhang"
TRIM_PM4_OPTIONS="-p 100000 -k 100"
TRIM_LCR_OPTIONS=""
TRIM_SR_OPTIONS=""
ASM_OVLP_OPTIONS=""
FSA_OL_FILTER_OPTIONS="--max_overhang=-1 --min_identity=-1"
FSA_ASSEMBLE_OPTIONS=""
CLEANUP=0

• Step 3: Correct Raw Reads. Correct the raw noisy reads using the following command:

$ mecat.pl correct ecoli_config_file.txt

• Step 4: Trim Out Low Quality Subsequences in Corrected Reads.

$ mecat.pl trim ecoli_config_file.txt

• Step 5: Assemble Contigs Using the Trimeed Reads

$ mecat.pl assemble ecoli_config_file.txt

• Step 6: Where to Find Results

  • The file ${MECAT_PATH}/eocli/ecoli/1-consensus/cns_reads_list.txt contains the full path of all corrected reads files.

   $ cat ${MECAT_PATH}/eocli/ecoli/1-consensus//cns_reads_list.txt
   /home/chenying/smrt_asm/ecoli/ecoli/1-consensus/cns_cns_dir/p00000000.cns.fasta

  • The extracted longest 30x (The number 30 is indidated by the CNS_OUTPUT_COVERAGE option in the config file) corrected reads used for trimming is ${MECAT_PATH}/ecoli/ecoli/1-consensus/cns_final.fasta.
  • The trimmed reads is ${MECAT_PATH}/ecoli/ecoli/2-trim_bases/trimReads.fasta
  • The assembled contigs is ${MECAT_PATH}/ecoli/ecoli/4-fsa/contigs.fasta

Input Format

The input to MECAT2 is indicated by the RAWREADS option in the config file. It must be a full path. MECAT2 supports several different input formats:

• H5 format. H5 file format must first be transfered to FASTA format with ${MECAT_PATH}/DEXTRACT/dextract. For example:

$ find pathto/raw_reads -name "*.bax.h5" -exec readlink -f {} \; > reads.fofn
$ while read line; do   dextract -v $line >> reads.fasta ; done <  reads.fofn

After transformation, proceed to one of the following input case.

• FASTA format.

RAWREADS=/Users/sysu/Desktop/files/programs/ecoli/pacbio/ecoli/raw_reads.fasta

Or FASTA format compressed in GNU Zip (gzip) format

RAWREADS=/Users/sysu/Desktop/files/programs/ecoli/pacbio/ecoli/raw_reads.fasta.gz

• FASTQ format

RAWREADS=/Users/sysu/Desktop/files/programs/ecoli/pacbio/ecoli/raw_reads.fastq

Or FASTQ format compressed in GNU Zip (gzip) format

RAWREADS=/Users/sysu/Desktop/files/programs/ecoli/pacbio/ecoli/raw_reads.fastq.gz

• List format A file indicates the full paths of all raw reads files.

RAWREADS=/Users/sysu/Desktop/files/programs/tomato/read_list.txt

$ cat /Users/sysu/Desktop/files/programs/tomato/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.

Program Descriptions

We describe in detail each module of MECAT, including their options and output formats.

Config File

MECAT2 reads all the information, including project name, raw reads, and various running parameters, from config file. To create a config file template, just run

$ mecat.pl config config_file_name

The above command creates a config file named config_file_name. We have met an sample of config file in the previous section

PROJECT=ecoli
RAWREADS=/home/chenying/smrt_asm/ecoli/ecoli_filtered.fastq
GENOME_SIZE=4800000
THREADS=4
MIN_READ_LENGTH=2000
CNS_OVLP_OPTIONS="-kmer_size 13"
CNS_PCAN_OPTIONS="-p 100000 -k 100"
CNS_OPTIONS=""
CNS_OUTPUT_COVERAGE=30
TRIM_OVLP_OPTIONS="-skip_overhang"
TRIM_PM4_OPTIONS="-p 100000 -k 100"
TRIM_LCR_OPTIONS=""
TRIM_SR_OPTIONS=""
ASM_OVLP_OPTIONS=""
FSA_OL_FILTER_OPTIONS="--max_overhang=-1 --min_identity=-1"
FSA_ASSEMBLE_OPTIONS=""
CLEANUP=0

The meaning of each option is given below

• PROJECT=ecoli, the name of the project. In this example, a directory ecoli will be created in the current directory, and then everything will take place in the directory ecoli.
• RAWREADS=, the raw reads (with full path) to be processed by MECAT2. See Input Format.
• GENOME_SIZE=, the size (in bp) of the underlying genome.
• THREADS=, number of CPU threads used by MECAT2.
• MIN_READ_LENGTH=, minimal length of corrected reads and trimmed reads.
• CNS_OVLP_OPTIONS="", options for detecting overlap candidates in the correction stage. Run mecat2map -help for details. Note that the output format is seqidx (-outfmt seqidx), which is set internally by mecat.pl.
• CNS_OPTIONS="", options for correcting raw reads. Run mecat2cns -help for details.
• TRIM_OVLP_OPTIONS="", options for detecting overlaps in the trimming stage. Run mecat2map for details. Note that output format is m4x (-outfmt m4x), which is set internally by mecat.pl.
• ASM_OVLP_OPTIONS="", options for detecting overlaps in the assemble stage. Run mecat2map -help for details. The output format is m4 (-outfmt m4), which is set internally by mecat.pl.
• FSA_OL_FILTER_OPTIONS="", options for filtering overlaps. See below for details.
• FSA_ASSEMBLE_OPTIONS="", options for assembling trimmed reads. See below for details.
• USE_GRID=false, using multiple computing nodes (true) or not (false).
• CLEANUP=0, delete intermediate date genrated by MECAT2 (1) or not (0). Please note the in assemblying large genomes, the intermediate data can be very large.
• CNS_OUTPUT_COVERAGE=30, number of coverage of the longest corrected reads are extracted to be trimed and then assembled. In this example, 30x (specifically, 30 * 4800000 = 144 MB) of the longest corrected reads will be extracted.

The MECAT2 Workflow.

For easy use, we have integrated all the procedures into one perl script file mecat.pl, which works in the following steps:

meat.pl config, as mentioned above, this command creates a config file. mecat.pl correct, correct raw reads, which consits of three steps:

detecting overlap candidates using mecat2map. partition overlap candidates into several parts using mecat2pcan. Each parts contains overlap candidates needed for correcting 100000 raw reads. correct raw reads based on overlap candidates using mecat2cns.

mecat.pl assemble, assemble corrected reads in three steps:

extract 30x longest corrected reads with mecat2extseqs trim out low quality subsequences in two stpes:

detecting overlaps of extracted reads using mecat2map trim out low quality subsequence based on their overlaps using mecat2lcr, mecat2splitreads and mecat2trimbases.

assemble trimmed reads into contigs in three steps:

detecting overlaps of trimmed reads using mecat2map filter out low quality overlaps using fsa_ol_filter assemble trimmed reads into contigs based on high quality overlaps using fsa_assemble

SMRT sequence alignment tool mecat2map

options

The command for running mecat2pw is

mecat2map [OPTIONS] reads reference > results.m4

Correction Tool mecat2cns

Overlap Filter fsa_ol_filter

fsa_ol_filter is used for filtering out low-quality overlaps. The usage of fsa_ol_filter1 is

fsa_ol_filter [optioins] overlaps filtered_overlaps

The options are

• --min_length=INT, minimum length of reads (default: 2500)
• --max_length=INT, maximum length of reads (defualt: INT_MAX).
• --min_identity=DOUBLE, minimum identity of overlaps (defualt: 90).
• --min_aligned_length=INT, minimum aligned length of overlaps (default: 2500).
• --max_overhang=INT, maximum overhang of overlaps (default: 10), negative number = determined by the program.
• --min_coverage=INT, minimum base coverage (default: -1), negative number = determined by the program.
• --max_coverage=INT, maximum base coverage (default: -1), negative number = determined by the program.
• --max_diff_coverage=INT, maximum difference of base coverage (default: -1), negative number = determined by the program.
• --coverage_discard=DOUBLE, discard ratio of base coverage (default: 0.01). If --max_coverage or --max_diff_coverage is negative, it will be reset to (100-coverage_discard)th percentile.
• --overlap_file_type="|m4|paf|ovl", overlap file format (default: ""). "" = filename extension, "m4" = M4 format, "paf" = PAF format generated by minimap2, "ovl" = OVL format generated by FALCON.
• --bestn=INT, output best n overlaps on 5' or 3' end for each read (default: 10).
• --genome_size=INT, genome size. It determines the maximum length of reads with --coverage together.
• --coverage=INT, coverage. It determines the maximum length of reads with --genome_size together.
• --output_directory=STRING, directory for output files (default: ".").
• --thread_size=INT, number of threads (default: 4).

Assemble Tool fsa_assemble

fsa_assemble is a tool for constructing contigs from filtered overlaps and corrected reads. The algorithm is similar to FALCON. The usage of fsa_assemble is

fsa_assenble [optioins] filtered_overlaps

The options are

• --min_length=INT, minimum length of reads (default: 0).
• --min_identity=DOUBLE, minimum identity of overlaps (defualt: 0).
• --min_aligned_length=INT, minimum aligned length of overlaps (default: 0).
• --min_contig_length=INT, minimum length of contigs (default: 500).
• --read_file=STRING, reads file name in FASTA or FASTQ format.
• --overlap_file_type="|m4|paf|ovl", overlap file format (default: ""). "" = filename extension, "m4" = M4 format, "paf" = PAF format generated by minimap2, "ovl" = OVL format generated by FALCON.
• --output_directory=STRING, directory for output files (default: ".").
• --select_branch="no|best", selecting method when encountering branches in the graph, "no" = do not select any branch, "best" = select the most probable branch.
• --thread_size=INT, number of threads (default: 4)

Citation

Chuan-Le Xiao, Ying Chen, Shang-Qian Xie, Kai-Ning Chen, Yan Wang, Yue Han, Feng Luo, Zhi Xie. MECAT: fast mapping, error correction, and de novo assembly for single-molecule sequencing reads. Nature Methods, 2017, 14: 1072-1074

Contact

• Chuan-Le Xiao, [email protected]

• Ying Chen, [email protected]

• Fan Nie, [email protected]

• Feng Luo, [email protected]

Update Information

Updates in MECAT2 (20193.14):

• Add some improvements in FSA

• Optimize Install Method

Updates in MECAT2 (2019.2):

• Fix many bugs in MECAT

• Replace the asseble module mecat2canu by fasa.

Updates in MECAT V1.3 (2017.12.18):

• Correct text error in HDF5 Installation.

• Update the makefile in dextract .

• Update citation.

Updates in MECAT V1.2 (2017.5.22):

• Add trimming module in mecat2canu to improve the integrality of the assembly.

• Add supports for Nanopore data.

• Improve the sensitivity of mecat2ref.

MECAT v1.1 replaced the old MECAT,some debug were resolved and some new fuctions were added:

• 1. we added the extracted tools for the raw H5 format files.
• 2. some debugs from running mecat2canu were solved