More at http://www.nabi.res.in/Vacancies/NABI/ResearchFellowships/JRFSRFRA/2016/NABI8(18)2012-PME-3/Advt.pdf

Again, running spades.py will show you the options:

spades.py

This produces:

SPAdes genome assembler v3.10.1

Usage: /usr/local/SPAdes-3.10.1-Linux/bin/spades.py [options] -o <output_dir>

Basic options:
-o      <output_dir>    directory to store all the resulting files (required)
--sc                    this flag is required for MDA (single-cell) data
--meta                  this flag is required for metagenomic sample data
--rna                   this flag is required for RNA-Seq data
--plasmid               runs plasmidSPAdes pipeline for plasmid detection
--iontorrent            this flag is required for IonTorrent data
--test                  runs SPAdes on toy dataset
-h/--help               prints this usage message
-v/--version            prints version

Input data:
--12    <filename>      file with interlaced forward and reverse paired-end reads
-1      <filename>      file with forward paired-end reads
-2      <filename>      file with reverse paired-end reads
-s      <filename>      file with unpaired reads
--pe<#>-12      <filename>      file with interlaced reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-1       <filename>      file with forward reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-2       <filename>      file with reverse reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-s       <filename>      file with unpaired reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-<or>    orientation of reads for paired-end library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--s<#>          <filename>      file with unpaired reads for single reads library number <#> (<#> = 1,2,..,9)
--mp<#>-12      <filename>      file with interlaced reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-1       <filename>      file with forward reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-2       <filename>      file with reverse reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-s       <filename>      file with unpaired reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-<or>    orientation of reads for mate-pair library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--hqmp<#>-12    <filename>      file with interlaced reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-1     <filename>      file with forward reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-2     <filename>      file with reverse reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-s     <filename>      file with unpaired reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-<or>  orientation of reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--nxmate<#>-1   <filename>      file with forward reads for Lucigen NxMate library number <#> (<#> = 1,2,..,9)
--nxmate<#>-2   <filename>      file with reverse reads for Lucigen NxMate library number <#> (<#> = 1,2,..,9)
--sanger        <filename>      file with Sanger reads
--pacbio        <filename>      file with PacBio reads
--nanopore      <filename>      file with Nanopore reads
--tslr  <filename>      file with TSLR-contigs
--trusted-contigs       <filename>      file with trusted contigs
--untrusted-contigs     <filename>      file with untrusted contigs

Pipeline options:
--only-error-correction runs only read error correction (without assembling)
--only-assembler        runs only assembling (without read error correction)
--careful               tries to reduce number of mismatches and short indels
--continue              continue run from the last available check-point
--restart-from  <cp>    restart run with updated options and from the specified check-point ('ec', 'as', 'k<int>', 'mc')
--disable-gzip-output   forces error correction not to compress the corrected reads
--disable-rr            disables repeat resolution stage of assembling

Advanced options:
--dataset       <filename>      file with dataset description in YAML format
-t/--threads    <int>           number of threads
                                [default: 16]
-m/--memory     <int>           RAM limit for SPAdes in Gb (terminates if exceeded)
                                [default: 250]
--tmp-dir       <dirname>       directory for temporary files
                                [default: <output_dir>/tmp]
-k              <int,int,...>   comma-separated list of k-mer sizes (must be odd and
                                less than 128) [default: 'auto']
--cov-cutoff    <float>         coverage cutoff value (a positive float number, or 'auto', or 'off') [default: 'off']
--phred-offset  <33 or 64>      PHRED quality offset in the input reads (33 or 64)
                                [default: auto-detect]

As you can see this is also a “pipeline” of tools that can be switched on or off. SPAdes takes quite a long time, so for the purposes of this practical, something like this may suffice:

spades.py -t 4 \
          -m 32 \
          -k 31,51,71 \
          --only-assembler \
          -1 miseq.1.fastq -2 miseq.2.fastq \
          --nanopore minion.fastq \
          -o hybrid_assembly

In turn, these parameters mean

• use 4 threads
• max memory is 32Gb
• use 3 kmer values to build the de bruijn graph(s) - 31, 51 and 71
• only run the assembler, not the correction algorithm (for speed)
• read 1 and read 2 of the MiSeq data
• the nanopore data
• put the output in folder “hybrid_assembly”

Following are the genome assembly tools based on string graph:

1.SGA (String Graph Assembler) https://github.com/jts/sga

Assembles large genomes from high coverage short read data. SGA is designed as a modular set of programs, which are used to form an assembly pipeline. SGA implements a set of assembly algorithms based on the FM-index. As the FM-index is a compressed data structure, the algorithms are very memory efficient. The SGA assembly has three distinct phases. The first phase corrects base calling errors in the reads. The second phase assembles contigs from the corrected reads. The third phase uses paired end and/or mate pair data to build scaffolds from the contigs. The output of this software is a PDF report that allows the properties of the genome and data quality to be visually explored. By providing more information to the user at the start of an assembly project, this software will help increase awareness of the factors that make a given assembly easy or difficult, assist in the selection of software and parameters and help to troubleshoot an assembly if it runs into problems.

2. SAGE: String-overlap Assembly of GEnomes https://github.com/lucian-ilie/SAGE2

SAGE, for de novo genome assembly. As opposed to most assemblers, which are de Bruijn graph based, SAGE uses the string-overlap graph. SAGE builds upon great existing work on string-overlap graph and maximum likelihood assembly, bringing an important number of new ideas, such as the efficient computation of the transitive reduction of the string overlap graph, the use of (generalized) edge multiplicity statistics for more accurate estimation of read copy counts, and the improved use of mate pairs and min-cost flow for supporting edge merging. The assemblies produced by SAGE for several short and medium-size genomes compared favourably with those of existing leading assemblers.

3. FSG: Fast String Graph

The new integrated assembler has been assessed on a standard benchmark, showing that fast string graph (FSG) is significantly faster than SGA while maintaining a moderate use of main memory, and showing practical advantages in running FSG on multiple threads. Moreover, we have studied the effect of coverage rates on the running times.

4.  BASE https://github.com/dhlbh/BASE

It enhances the classic seed-extension approach by indexing the reads efficiently to generate adaptive seeds that have high probability to appear uniquely in the genome. Such seeds form the basis for BASE to build extension trees and then to use reverse validation to remove the branches based on read coverage and paired-end information, resulting in high-quality consensus sequences of reads sharing the seeds. Such consensus sequences are then extended to contigs. BASE is a practically efficient tool for constructing contig, with significant improvement in quality for long NGS reads. It is relatively easy to extend BASE to include scaffolding.

5. Fermi https://github.com/lh3/fermi/

Fermi is a de novo assembler with a particular focus on assembling Illumina short sequence reads from a mammal-sized genome. In addition to the role of a typical assembler, fermi also aims to preserve heterozygotes which are often collapsed by other assemblers. Its ultimate goal is to find a minimal set of unitigs to represent all the information in raw reads.

If you want to learn about String Graph assembler, please read the following papers -

i) The Fragment Assembly String Graph - E. W. Myers

This paper describes the String Graph concept.

ii) Efficient construction of an assembly string graph using the FM-index - Jared T. Simpson and Richard Durbin

This earlier paper from Simpson and Durbin

iii) Efficient de novo assembly of large genomes using compressed data structures - Jared T. Simpson and Richard Durbin

More at http://www.du.ac.in/du/index.php?mact=News,cntnt01,detail,0&cntnt01articleid=12859&cntnt01returnid=83

The tool should be compatible with most UNIX flavors and has been successfully tested on the following operating systems:

• Mac OS X 10.11.1
• Mac OS X 10.10.3
• Ubuntu 14.04 LTS

Address of the bookmark: https://github.com/mculinovic/EAGLER

Address of the bookmark: http://wishart.biology.ualberta.ca/cgview/

If you have any questions or comments, post them on one of the trackers on BRIG’s SourceForge page: http://sourceforge.net/tracker/?group_id=328245.

Features:

• Images show similarity between a central reference sequence and other sequences as concentric rings.
• BRIG will perform all BLAST comparisons and file parsing automatically via a simple GUI.
• Contig boundaries and read coverage can be displayed for draft genomes; customized graphs and annotations can be displayed.
• Using a user-defined set of genes as input, BRIG can display gene presence, absence, truncation or sequence variation in a set of complete genomes, draft genomes or even raw, unassembled sequence data.
• BRIG also accepts SAM-formatted read-mapping files enabling genomic regions present in unassembled sequence data from multiple samples to be compared simultaneously

Address of the bookmark: http://brig.sourceforge.net/

AVAILABILITY AND IMPLEMENTATION:

http://cab.spbu.ru/software/quast-lg

Address of the bookmark: http://cab.spbu.ru/software/quast-lg/

https://academic.oup.com/bioinformatics/article/31/10/1674/177884

Address of the bookmark: https://github.com/voutcn/megahit

You see, for the past couple of years, I’ve been a bit frustrated because OS X does not come with a whole lot of Perl modules pre-installed, and for all I googled, I couldn’t find an “idiot’s” guide for moderately-savvy-but-not-expert users like myself to install modules and dependencies on demand.

The only instructions I could find point to Fink, which basically installs modules in a path that isn’t included in the Perl @INC variable, meaning you have to manually specify the full path to the modules in every script — which is not a lot of fun if you’re developing on OS X and deploying on Red Hat, for instance.

Moreover, Fink doesn’t seem to make every module available, and it’s not very easy to determine which Fink package you need to install if you need a particular module.

So, with a script that called on several apparently unavailable modules, and a deadline looming, I finally decided to suck it up and figure out how to use CPAN to install them:

1) Make sure you have the Apple Developer Tools (XCode) installed.

These are on one of your install discs, or available as a huge but free download from the Apple Developer Connection [free registration required] or the Mac App Store. I thought I had them, but apparently when we upgraded that computer to Tiger, they went missing.

If you don’t have this stuff installed, your installation will fail with errors about unavailable commands.

1.5) Install Command Line Tools (Recent XCode versions only)

(Thank you to Tom Marchioro for informing me about this step.)

Older versions of XCode installed the command line tools (which are required to properly install CPAN modules) by default, but apparently newer ones do not. To check whether you have the command line tools already installed, run the following from the Terminal:

$ which make

This command checks the system for the “make” tool. If it spits out something like /usr/bin/make you’re golden and can skip ahead to Step 2. If you just get a new prompt and no output, you’ll need to install the tools:

1. Launch XCode and bring up the Preferences panel.
2. Click on the Downloads tab
3. Click to install the Command Line Tools

If you like, you can run which make again to confirm that everything’s installed correctly.

2) Configure CPAN.

$ sudo perl -MCPAN -e shell

perl> o conf init

This will prompt you for some settings. You can accept the defaults for almost everything (just hit “return”). The two things you must fill in are the path to make (which should be /usr/bin/make or the value returned when you run which make from the command line) and your choice of CPAN mirrors (which you actually choose don’t really matter, but it won’t let you finish until you select at least one). If you use a proxy or a very restrictive firewall, you may have to configure those settings as well.

If you skip Step 2, you may get errors about make being unavailable.

3) Upgrade CPAN

$ sudo perl -MCPAN -e 'install Bundle::CPAN'

Don’t forget the sudo, or it’ll fail with permissions errors, probably when doing something relatively unimportant like installing man files.

This will spend a long time downloading, testing, and compiling various files and dependencies. Bear with it. It will prompt you a few times about dependencies. You probably want to enter “yes”. I agreed to everything it asked me, and everything turned out fine. YMMV of course. If everything installs properly, it’ll give you an “OK” at the end.

4) Install your modules. For each module….

$ sudo perl -MCPAN -e 'install Bundle::Name'

or

$ sudo perl -MCPAN -e 'install Module::Name'

This will install the module and its dependencies. Nice, eh? Again, don’t forget the sudo.

The first time you run this after upgrading CPAN, it may prompt you to configure again (see Step 2). If you accept its offer to try to configure itself automatically, it may just run through everything without a problem.

There are a couple of potential pitfalls with specific modules (such as theLWP::UserAgent / HEAD issue), but most have workarounds, and I haven’t run into anything that wasn’t easily recoverable.

And that’s it!

Did you find this useful? Is there anything I missed?