instagraal 0.1.3

Large genome reassembly based on Hi-C data.

instaGRAAL

Large genome reassembly based on Hi-C data (continuation and partial rewrite of GRAAL) and post-scaffolding polishing libraries.

This work is under continuous development/improvement - see GRAAL for information about the basic principles.

Installation

   sudo pip3 install -e git+https://github.com/koszullab/instagraal.git@master#egg=instagraal

Requirements

The scaffolder and polishing libraries are written in Python 3 and CUDA. The Python 2 version is available at the python2 branch of this repository, but be aware that development will mainly focus on the Python 3 version. The software has been tested for Ubuntu 17.04 and most dependencies can be downloaded with its package manager (or Python's pip).

External libraries

You will need to download and install the NVIDIA CUDA toolkit. Manual installation is recommended - installing nvidia-cuda-toolkit from Ubuntu's package manager has been known to cause glitches.

OpenGL libraries:

• libglu1-mesa
• libxi-dev
• libxmu-dev
• libglu1-mesa-dev

HDF5 serialization library:

• hdf5-tools

Boost libraries:

• libboost-all-dev

Python libraries

• numpy
• scipy
• matplotlib
• codepy
• h5py
• pyopengl
• docopt
• biopython

These should be handily installed using the supplied requirements file:

pip3 install -r requirements.txt

You will also need to build pycuda with OpenGL support and disable its use of custom Boost libraries.

You may run (as root) instagraal-setup, an all-in-one script to handle all the above dependencies on Ubuntu 17+.

How to use

Unlike GRAAL, this is meant to be run from the command line.

Usage

instagraal <hic_folder> <reference.fa> [<output_folder>]
           [--level=4] [--cycles=100] [--coverage-std=1]
           [--neighborhood=5] [--device=0] [--circular] [--bomb]
           [--save-matrix] [--pyramid-only] [--save-pickle]
           [--quiet] [--debug]

Options

-h, --help              Display this help message.
--version               Display the program's current version.
-l 4, --level 4         Level (resolution) of the contact map.
                        Increasing level by one means a threefold smaller
                        resolution but also a threefold faster computation
                        time. [default: 4]
-n 100, --cycles 100    Number of iterations to perform for each bin.
                        (row/column of the contact map). A high number of
                        cycles has diminishing returns but there is a
                        necessary minimum for assembly convergence.
                        [default: 100]
-c 1, --coverage-std 1  Number of standard deviations below the mean.
                        coverage, below which fragments should be filtered
                        out prior to binning. [default: 1]
-N 5, --neighborhood 5  Number of neighbors to sample for potential
                        mutations for each bin. [default: 5]
--device 0              If multiple graphic cards are available, select
                        a specific device (numbered from 0). [default: 0]
-C, --circular          Indicates genome is circular. [default: False]
-b, --bomb              Explode the genome prior to scaffolding.
                        [default: False]
--pyramid-only          Only build multi-resolution contact maps (pyramids)
                        and don't do any scaffolding. [default: False]
--save-pickle           Dump all info from the instaGRAAL run into a
                        pickle. Primarily for development purposes, but
                        also for advanced post hoc introspection.
                        [default: False]
--save-matrix           Saves a preview of the contact map after each
                        cycle. [default: False]
--quiet                 Only display warnings and errors as outputs.
                        [default: False]
--debug                 Display debug information. For development purposes
                        only. Mutually exclusive with --quiet, and will
                        override it. [default: False]

Input datasets

The above <hic_folder> passed as an argument to instaGRAAL needs three files:

• A file named abs_fragments_contacts_weighted.txt, containing the (sparse) Hi-C map itself. The first line must be id_frag_a id_frag_b n_contact. All subsequent lines must represent the map's contacts in coordinate format (id_frag_a being the row indices, id_frag_b being the column indices, n_contact being the number of contacts between each locus or index pair, e.g. if 5 contacts are found between fragments #2 and #3, there should be a line reading 2 3 5 in the file). n_contact must be an integer. The list should be sorted according to id_frag_a first, then id_frag_b. Fragment ids start at 0.
• A file named fragments_list.txt containing information related to each fragment of the genome. The first line must be id chrom start_pos end_pos size gc_content, and subsequent lines (representing the fragments themselves) should follow that template. The fields should be self-explanatory; notably, chrom can be any string representing the chromosome's name to which the fragment at a given line belongs, and fragment ids should start over at 1 when the chromosome name changes. Aside from the chrom field and the gc field which is currently unused in this version and can be filled with any value, all fields should be integers. Note that start_pos starts at 0.
• A file named info_contigs.txt containing information related to each contig/scaffold/chromosome in the genome. The first line must be contig length_kb n_frags cumul_length. Field names should be again self-explanatory; naturally the contig field must contain names that are consistent with those found in fragments_list.txt. Also length_kb should be an integer (rounded up or down if need be), and n_frags and cumul_length are supposed to be consistent with each other in that the cumulated length (in fragments) of contig N should be equal to the sum of the fields found in n_frags for the N-1 preceding lines. Note that cumul_length starts at 0.

All fields (including those in the files' headers) must be separated by tabs.

Minimal working templates are provided in the example folder.

Polishing

Lingering artifacts found in output genomes can be corrected by editing the info_frags.txt file, either by hand or with a script. Look at options by running the following:

instagraal-polish -h

The most common use case is to run all polishing procedures at once:

instagraal-polish -m polishing -i info_frags.txt -f reference.fasta -o polished_assembly.fa

Troubleshooting

Loading CUDA libraries

If you encounter the following error, despite having installed the NVIDIA CUDA Toolkit:

ImportError: libcurand.so.9.2: cannot open shared object file: No such file or directory

it probably means the CUDA-related libraries haven't been properly added to your $PATH for some reason. A quick solution is to simply add this at the end of your .bashrc or .bash_profile (replace the paths with wherever you installed the toolkit and change the version number accordingly):

export PATH=/usr/local/cuda-9.2/bin${PATH:+:${PATH}}
export LD_LIBRARY_PATH=/usr/local/cuda-9.2/lib64${LD_LIBRARY_PATH:+:${LD_LIBRARY_PATH}}

Remote running

If you encounter the following error:

freeglut (main_single_proc.py): failed to open display ''

it most likely means you attempted to run an instaGRAAL instance remotely (e.g. over ssh) but didn't configure a proper $DISPLAY variable. In order to avoid this, simply run the following beforehand:

export DISPLAY=:0

Note that this will disable the movie (it will play on the remote machine instead).

However, instaGRAAL is based on OpenGL, which means there has to be an X server of some kind running on your target machine no matter what. While this allows for pretty movies and visualizations, it may prove problematic on an environment you don't have total control over, e.g. a server cluster. Currently, your best bet is asking the system administrator of the target machine to set up an X instance if they haven't already.

References

Principle

• High-quality genome assembly using chromosomal contact data, Hervé Marie-Nelly, Martial Marbouty, Axel Cournac, Jean-François Flot, Gianni Liti, Dante Poggi Parodi, Sylvie Syan, Nancy Guillén, Antoine Margeot, Christophe Zimmer and Romain Koszul, Nature Communications, 2014
• A probabilistic approach for genome assembly from high-throughput chromosome conformation capture data, Hervé Marie-Nelly, 2013, PhD thesis

Use cases

• Proximity ligation scaffolding and comparison of two Trichoderma reesei strains genomes, Etienne Jourdier, Lyam Baudry, Dante Poggi-Parodi, Yoan Vicq, Romain Koszul, Antoine Margeot, Martial Marbouty, and Frédérique Bidard, Biotechnology for Biofuels, 2017
• Scaffolding bacterial genomes and probing host-virus interactions in gut microbiome by proximity ligation (chromosome capture) assay, Martial Marbouty, Lyam Baudry, Axel Cournac, and Romain Koszul, Science Advances, 2017

Contact

• [e-mail]
• [e-mail]