MetAssemble
===========

Content
1. Overview
2. Dependencies
3. Usage
A. Assembly Output List


1. Overview
===========

MetAssemble is a pipeline that runs several metagenomic assembly strategies
combining Velvet, Meta-Velvet, Minimus2 and Bambus2 on Illumina paired end
reads. The pipeline was originally developed to validate the performance of the
individual strategies, but can be used to perform the assembly strategies
without validation as well.  The pipeline is written in GNU Make and consists
of two parts, the first part performs the various assembly strategies
(Makefile). The second part validates the assemblies done in the first part
(Makefile-validate-assemblies). Currently the code works specifically in
combination with sbatch to launch jobs with correct dependencies and resource
allocations on our computing facility.  With a little tweaking the code can be
used on another system that uses sbatch (adjust the partition names and the
account used to schedule the job). The pipeline also works out of the box
without sbatch but do note that running all the assemblies can take a very long
time in that case so please specify the number of jobs with 'make -n' to gain
some speed-up.

2. Dependencies
===============

Supported input:
- Illumina fastq CASAVA v1.8 paired end reads

-- Makefile --
Features four steps of the metagenomic assembly pipeline:
1) Read processing.
- Quality trimming
    * Trim.pl
    http://wiki.bioinformatics.ucdavis.edu/index.php/Trim.pl
2) Assembling contigs
- De Bruijn graph
    * Velvet 1.2.01
    http://www.ebi.ac.uk/~zerbino/velvet/
    * Meta-Velvet 1.2.01
    http://metavelvet.dna.bio.keio.ac.jp/
3) Merging contigs
- With cd-hit and minimus2
http://ged.msu.edu/angus/metag-assembly-2011/velvet-multik.html
    * cd-hit-est
    http://weizhong-lab.ucsd.edu/cd-hit/
    * Minimus2 from Amos
    http://sourceforge.net/apps/mediawiki/amos/index.php?title=Minimus2
- Newbler RunAssembly 2.6 
4) Scaffolding
- Construct linkage information by mapping reads to contigs
    * BWA
    http://bio-bwa.sourceforge.net/
- Scaffold contigs
    * Bambus2
    http://sourceforge.net/apps/mediawiki/amos/index.php?title=Bambus2

-- Makefile-validate-assemblies --
- Mapping contigs to reference
    * MUMmer 3.23
    http://sourceforge.net/projects/mummer/files/

3. Usage
========

To run all assemblies copy Makefile to the directory of the Illumina paired end
reads. Adjust the four input parameters in the Makefile or pass them as
arguments:

# Input parameters
FASTQ1=500pg_even_GGACTCCT-CTCTCTAT_L003_R1_001.fastq
FASTQ2=500pg_even_GGACTCCT-CTCTCTAT_L003_R2_001.fastq
FASTQBASE=500pg_even
SCRIPTDIR=/bubo/home/h16/inod/glob/github/metassemble/scripts

Output parameters can be adjusted as well, by default everything is outputted
to ./ma-out/. Assemblies are in ./ma-out/assemblies and the quality trimmed
reads are in ma-out/processed-reads. If your system uses sbatch to schedule
jobs, change USE_SBATCH to yes and change account, userid and partition
parameters accordingly. The node partition in the code has 24GB memory and 8
cores, the core partition, one core and 3 GB. The set allocations worked for
all libraries smaller than 12M pairs (24M reads total). Resource allocation is
static, not based on input file sizes.

After setting input parameters and possibly sbatch parameters. Running 'make
all' will run all assemblies. They can be found in section A. In case one uses
sbatch the Bambus2 assemblies have to be run separately afterwards with
'make bambus2'. If sbatch is not used, use 'make -j <nrofjobs>' and specify the
number of jobs to run simultaneously to gain some speed-up.

The output of all the assemblies are relative to ma-out/assemblies/ and follow
this structure:

velvet/noscaf/noscaf_%/contigs.fa                                     
# velvetg -scaffolding no and % is the kmer size
velvet/scaf/scaf_%/contigs.fa                                         
# velvetg -scaffolding yes and % is the kmer size
metavelvet/noscaf/noscaf_%/meta-velvetg.contigs.fa                    
# meta-velvetg -scaffolding no and % is the kmer size
metavelvet/scaf/scaf_%/meta-velvetg.contigs.fa                        
# meta-velvetg -scaffolding yes and % is the kmer size
velvet/noscaf/minimus2/all-merged.fasta                               
# Minimus2 merged velvet noscaf assembly
velvet/noscaf/newbler/454AllContigs.fna                               
# Newbler merged velvet noscaf assembly
metavelvet/noscaf/minimus2/all-merged.fasta                           
# Minimus2 merged metavelvet noscaf assembly
metavelvet/noscaf/newbler/454AllContigs.fna                           
# Newbler merged metavelvet noscaf assembly
velvet/noscaf/noscaf_%/bambus2/bambus2.scaffold.linear.fasta          
# Bambus 2 scaffolding of velvetg -scaffolding no and % is kmer size
metavelvet/noscaf/noscaf_%/bambus2/bambus2.scaffold.linear.fasta      
# Bambus 2 scaffolding of meta-velvetg -scaffoldng no and % is kmer size
velvet/noscaf/minimus2/bambus2/bambus2.scaffold.linear.fasta          
# Bambus2 scaffolding of minimus2 merged velvet no scaf assemblies
velvet/noscaf/newbler/bambus2/bambus2.scaffold.linear.fasta           
# Bambus2 scaffolding of newbler merged velvet no scaf assemblies

NOTE: kmer size between 31 and 84, change Makefile for larger or smaller kmers

One assembly can be done with:
make /path/to/assembly/assembly.fasta
For instance:
make ma-out/assemblies/velvet/noscaf/noscaf_31/contigs.fa
To only get the commands for a specific assembly type:
make -Bn /path/to/assembly/assembly.fasta
A full listing of all assemblies can be found below.

* TODO: Usage for Makefile-validate-assemblies


A. Assembly output listing
==========================
ma-out/assemblies/velvet/noscaf/noscaf_31/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_33/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_35/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_37/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_39/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_41/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_43/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_45/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_47/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_49/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_51/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_53/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_55/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_57/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_59/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_61/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_63/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_65/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_67/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_69/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_71/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_73/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_75/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_77/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_79/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_81/contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_83/contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_31/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_33/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_35/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_37/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_39/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_41/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_43/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_45/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_47/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_49/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_51/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_53/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_55/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_57/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_59/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_61/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_63/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_65/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_67/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_69/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_71/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_73/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_75/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_77/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_79/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_81/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/noscaf/noscaf_83/meta-velvetg.contigs.fa
ma-out/assemblies/velvet/noscaf/minimus2/all-merged.fasta
ma-out/assemblies/metavelvet/noscaf/minimus2/all-merged.fasta
ma-out/assemblies/velvet/noscaf/newbler/454AllContigs.fna
ma-out/assemblies/metavelvet/noscaf/newbler/454AllContigs.fna
ma-out/assemblies/velvet/scaf/scaf_31/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_33/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_35/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_37/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_39/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_41/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_43/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_45/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_47/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_49/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_51/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_53/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_55/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_57/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_59/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_61/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_63/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_65/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_67/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_69/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_71/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_73/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_75/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_77/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_79/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_81/contigs.fa
ma-out/assemblies/velvet/scaf/scaf_83/contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_31/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_33/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_35/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_37/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_39/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_41/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_43/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_45/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_47/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_49/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_51/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_53/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_55/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_57/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_59/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_61/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_63/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_65/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_67/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_69/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_71/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_73/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_75/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_77/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_79/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_81/meta-velvetg.contigs.fa
ma-out/assemblies/metavelvet/scaf/scaf_83/meta-velvetg.contigs.fa
ma-out/assemblies/velvet/noscaf/noscaf_31/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_33/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_35/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_37/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_39/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_41/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_43/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_45/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_47/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_49/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_51/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_53/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_55/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_57/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_59/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_61/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_63/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_65/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_67/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_69/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_71/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_73/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_75/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_77/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_79/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_81/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/noscaf_83/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_31/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_33/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_35/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_37/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_39/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_41/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_43/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_45/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_47/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_49/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_51/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_53/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_55/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_57/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_59/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_61/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_63/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_65/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_67/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_69/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_71/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_73/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_75/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_77/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_79/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_81/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/noscaf_83/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/minimus2/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/minimus2/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/velvet/noscaf/newbler/bambus2/bambus2.scaffold.linear.fasta
ma-out/assemblies/metavelvet/noscaf/newbler/bambus2/bambus2.scaffold.linear.fasta