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LACHESIS User's Manual and Quick Start Guide

LACHESIS: A software tool to measure the thread of life.

Created by Josh Burton (jnburton at uw.edu) in the Department of Genome Sciences at the University of Washington, Seattle, WA, USA

Publication in Nature Biotechnology (please cite) is here: http://dx.doi.org/10.1038/nbt.2727

Table of Contents

INSTALLING LACHESIS
  1. System requirements
  2. Downloading the LACHESIS package
  3. Compiling the LACHESIS package
  4. Testing out LACHESIS on a sample dataset
RUNNING LACHESIS
  1. Input requirements
  2. Creating an INI file
  3. Aligning the Hi-C reads to the draft assembly
  4. Filtering the Hi-C reads
  5. Aligning the draft assembly to the reference assembly, if there is one
  6. Running LACHESIS
  7. Interpreting the LACHESIS results
TROUBLESHOOTING
  1. LACHESIS won't compile!
  2. LACHESIS is crashing!
  3. LACHESIS is producing a weird result!
COPYRIGHT AND DISCLAIMER
ACKNOWLEDGMENTS

Installing LACHESIS

1. System requirements

To setup and run LACHESIS, you will need a computer running in a UNIX environment with at least 16GB of memory, with the following software installed:

Note that LACHESIS requires a minimum stack size of 10MB (check with ulimit -s). If your system uses 8MB (e.g. Fedora or Ubuntu), you will need to increase the stack size (ulimit -s 10240).

You may also need the following software:

2. Downloading the LACHESIS package

Download the LACHESIS package from http://shendurelab.github.io/LACHESIS/ into a UNIX filesystem. If you download the tarball (LACHESIS.tar.gz), unpack it using the following UNIX commands:

tar xzvf LACHESIS.tar.gz
cd LACHESIS/

3. Compiling the LACHESIS package

To compile LACHESIS, you must first download and install two other libraries: boost (available at http://www.boost.org/) and SAMtools (available at http://samtools.sourceforge.net/). Once these are installed, set the shell environment variables $LACHESIS_BOOST_DIR and $LACHESIS_SAMTOOLS_DIR to point to the directories containing these packages. The command for setting an environment variable will depend on what Unix shell you are using. For example, in bash, type: export LACHESIS_BOOST_DIR=/path/to/boost/ and export LACHESIS_SAMTOOLS_DIR=/path/to/samtools/ (replacing the paths with the paths where you install the libraries.) (LACHESIS_BOOST_DIR should have the subdirectory stage/lib/.)

Finally, to compile LACHESIS, simply type make in the main LACHESIS directory.

4. Testing out LACHESIS on a sample dataset

This LACHESIS distribution includes a small sample dataset - specifically, a reduced version of the hESC dataset we used in our paper - that allows you to try out running LACHESIS. To run it, go into the LACHESIS directory and type Lachesis INIs/test_case.ini. The file test_case.ini gives the parameters that control how LACHESIS is run, in this case including the input files from the sample dataset. On my computer, LACHESIS takes 3 minutes to run on this dataset, most of which is in file I/O.

A directory called out/test_case/ will be created and will contain the results from this run. A summary of the results is in the file REPORT.txt; the main output files are in the subdirectory main_results/; other intermediate results are in the subdirectory cached_data/. The results from this test case won't be very good because the dataset of Hi-C links is so small, but they should give you an idea of how to run LACHESIS and what to expect from it.

Running Lachesis

1. Input requirements

So you want to use LACHESIS to scaffold your de novo assembly. You must start with two (or three) input files:

  • Your Hi-C reads, in an alignable format
  • Your draft de novo assembly, in fasta format
  • (Optional) A reference assembly, in fasta format

If you are going to use BWA to align your Hi-C reads, then the reads must be in a set of fastq or BAM files, and there should be two files for each library: one for the first read in each pair, and one for the second. If you are using another aligner, you may have your Hi-C reads in another format as required by that aligner.

Obviously you can't supply a reference assembly if no such assembly exists yet. But if there is one, the reference assembly can be input to LACHESIS in order to provide some very useful reference-based evaluation of the result. Even a partial sequence assembly or an assembly of a related species can be helpful. However, note that Lachesis is not an ideal piece of software for performing assisted assembly - that is, a semi-de novo assembly process that relies on the assumption of synteny with an existing reference genome. For more information on assisted assembly, see Gnerre et al, 2009.

2. Creating an INI file

Before running LACHESIS, you will need to create an initialization file, or INI file (*.ini). LACHESIS will parse this file and determine everything it needs to know in order to run, including the locations of all other input files. To create an INI file, make a copy of any of the INI files in the subdirectory INIs/ and edit that copy. The INI file contains several parameters; you will want to read through its documentation and set all parameters as you see fit.

Five of the INI parameters describe files that LACHESIS uses: DRAFT_ASSEMBLY_FASTA, SAM_FILES (in SAM_DIR), RE_SITE_SEQ, BLAST_FILE_HEAD, and REF_ASSEMBLY_FASTA. These all refer to files that you will need to create before running LACHESIS, by processing your input datasets. See the included file Data Prep Flowchart.png for a visual guide to this process.

Many of the other INI parameters provide heuristic parameters. It's worth your time to examine these parameters to get a better idea of what LACHESIS is doing. But if you're unsure of any of them, feel free to use the values in INIs/test_case.ini as defaults.

3. Aligning the Hi-C reads to the draft assembly

LACHESIS scaffolds de novo assemblies using the locations of the Hi-C reads on the assembly contigs/scaffolds. Hence you must align the Hi-C reads to the assembly contigs/scaffolds. You must ultimately produce files containing paired-read alignments in the SAM/BAM format (either SAMs or BAMs are acceptable; see http://samtools.sourceforge.net/). For each library of Hi-C reads (represented by a pair of fastq files) you should create a single SAM/BAM file describing the read pairs. DO NOT create a pair of SAM/BAM files, one for each read in the pair!

Aligning is a computationally intensive process and will take far more CPU time than LACHESIS itself will. You can use any aligner that produces SAM or BAM files, but we recommend BWA (http://bio-bwa.sourceforge.net/), which we used during the development of LACHESIS. If you use BWA, you will need to have your Hi-C reads in fastq format as described above, and you will need to perform the following steps:

  • Use bwa index to index your draft assembly. If your genome is large (>10 Mb), you will need to use the flag -a bwtsw. This will create a file <assembly>.fasta.bwt.
  • Use bwa aln to align the Hi-C reads to the indexed assembly. Each individual fastq file is aligned separately.
  • Use bwa sampe to determine the optimal placement of each read pair. This is where the two fastq files from each library are combined. The output of bwa sampe will be a SAM file (*.sam). You can compress this file to a BAM file (*.bam) using samtools (samtools view -bS <sam-file>) which will use less disk space and less I/O time.

4. Filtering the Hi-C reads

Many of the Hi-C reads can be determined to be noise, rather than signal. It is not strictly necessary to remove these reads from your BAM files before running LACHESIS. But it will reduce the files' size, reduce the I/O time required by LACHESIS to read them in, and may reduce LACHESIS' error rate.

We have included the scripts PreprocessSAMs.pl and PreprocessSAMs.sh, which perform the filtering methods we used. PreprocessSAMs.pl removes all reads that do not map within 500bp of a restriction enzyme site, following the suggestion of Yaffe & Tanay (Nature Genetics, 2011). To do this, it uses the script make_BED_around_RE_site.pl (also included) and also the bedtools library. PreprocessSAMs.pl also removes unpaired reads, which LACHESIS cannot use. Make sure to set the variable $RE_site in PreprocessSAMs.pl. You can also use the script PreprocessSAMs.sh as a batch script to run PreprocessSAMs.pl on a set of SAM/BAM files (set the variables SAMs and ASSEMBLY). The final set of SAM/BAM files is specified in the INI file as the parameter SAM_FILES, which names the files, and SAM_DIR, which names the directory they are in.

5. Aligning the draft assembly to the reference assembly, if there is one

You can skip this step if you aren't using a reference assembly. LACHESIS needs to know where the contigs/scaffolds from the draft assembly belong on the reference chromosomes. It can process the text output from the blastn command-line program, which you can read about and download at http://www.ncbi.nlm.nih.gov/books/NBK1763/. You will need to perform the following steps:

  • Create a BLAST database for the reference assembly: makeblastdb -in <ref-fasta> -dbtype=nucl -out <ref-fasta>.blastdb
  • Run blastn. Make sure you use the argument 'outfmt -7' so that blastn produces output in the format that LACHESIS expects.
  • If you have a large (mammalian-scale) draft assembly, blastn will take a long time to align it. You may want to split it up into separate fasta files and run blastn on each file separately. To see how we did this on the human assembly, see the included scripts blast.sh and blast.qsub.sh and the output files in test_case/draft_assembly/. The blastn output files from each chunk should be named <BLAST_FILE_HEAD>.NNN.blast.out, where NNN = 1, 2, 3, ...
  • The blastn output file(s) are specified in the INI file by the parameter BLAST_FILE_HEAD. Note that LACHESIS parses the BLAST output the first time it runs, then creates a cached file so that it doesn't have to take the time to parse the BLAST output again. This means that if you change the set of BLAST files, you'll need to delete the file at <OUTPUT_DIR>/cached_data/TrueMapping.assembly.txt in order to get LACHESIS to load your new set of BLAST files.

6. Running LACHESIS

If you've done steps 2-5 above, and you've created an INI file with the appropriate input file names, you should be good to go. Try running LACHESIS with the command: Lachesis <your-INI-file>. If there are any problems with the INI file, including missing input files, LACHESIS will immediately abort and will explain what went wrong. If not, then LACHESIS will begin loading in files, and all you need to do is wait and see what it does.

There are many parameters in the INI file beyond the inputs to LACHESIS. You may need to tweak some of these parameters in order to get an optimal assembly. Be sure to read the documentation in the INI file carefully so that you know what you're doing.

7. Interpreting the LACHESIS results

LACHESIS will create a set of output files in the directory OUTPUT_DIR that you have specified in the INI file. This directory contains a file REPORT.txt that will give a topline summary of LACHESIS' performance. It also contains two subdirectories, main_results/ and cached_data/. The main_results/ directory will contain the following files:

  1. clusters.txt and clusters.by_name.txt: These files indicate the clustering results. Each of LACHESIS' chromosome group is shown as a line, and the input contigs/scaffolds in that group are listed on the line, either by ID (clusters.txt) or by contig name (clusters.by_name.txt).
  2. group*.ordering: These files indicate the ordering and orienting results. There is one file for each group. In each file is a list of input contigs/scaffolds in order, with their orientations, orientation quality scores, and gap sizes.

To create the final assembly fasta, run the included script CreateScaffoldedFasta.pl. After you have run this, OUTPUT_DIR will contain the file Lachesis_assembly.fasta. This fasta file will contain your output assembly, in three sections of successive contigs:

  1. One large scaffold for each of the ordered and oriented chromosome groups predicted by LACHESIS (i.e., each of the group*.ordering files.)
  2. All of the input contigs/scaffolds that have been clustered into a chromosome group by LACHESIS, but were not ordered within that group. They will be given names indicating what chromosome group they belong in.
  3. All of the input contigs/scaffolds that were not clustered at all by LACHESIS.

TROUBLESHOOTING

LACHESIS is a good piece of software, but it isn't perfect. You may run it and get a result you weren't expecting. You may also run it and get no result at all because it crashes.

1. LACHESIS won't compile!

There are several reasons why LACHESIS may fail to compile. Some of the most common problems involve linking in the samtools and boost dependencies so that the LACHESIS source code can find them. You will need to set the environment variables LACHESIS_BOOST_DIR and LACHESIS_SAMTOOLS_DIR, and you should make sure you're using an older version of samtools (0.1.19 or earlier). For more details, see "Compiling the LACHESIS package", above.

2. LACHESIS is crashing!

If LACHESIS crashes, the first thing you should do is look carefully at its output. It might give a verbose explanation of what went wrong and give you a good idea for how to fix it. You may also receive an "assertion error", which looks like this: Assertion ... failed. That means that at some stage of the algorithm, LACHESIS encountered something specific that it wasn't expecting. An assertion error will come with a reference to the file (*.cc or *.h) and the line number where the error occurred. Try looking at that line in the file, which should contain the function assert(). There should be some comments around that line that explain what might be causing the assertion error.

If you run LACHESIS on the provided test case and a segmentation fault occurs, you're running into a known problem: a limitation in stack size (a low-level operating system attribute.) Some OS's (including Fedora and Ubuntu) set a default stack size of 8 MB, but LACHESIS needs 10 MB. To fix this, type: ulimit -s 10240

In general, we've made a strong effort to make LACHESIS a well-designed and well-commented piece of code. If you're familiar with C++, you should be able to poke around in the source code and get an idea of what's going on. We recommend starting with the top-level module, Lachesis.cc, and working from there.

If all else fails, and you still need help running LACHESIS, please e-mail Josh Burton at jnburton at uw.edu.

3. LACHESIS is producing a weird result!

After you've gotten LACHESIS to run properly, take a good look at the REPORT.txt file. If you're getting a weird result - for example, very little sequence is being assembled, or the error rate is high - you may need to tune LACHESIS' performance.

There are several heuristic parameters involved in the running of LACHESIS. They include: CLUSTER_MIN_RE_SITES, CLUSTER_MAX_LINK_DENSITY, CLUSTER_DO_NONINFORMATIVE, ORDER_MIN_N_RES_IN_TRUNK, and ORDER_MIN_N_RES_IN_SHREDS. These parameters are tuning knobs that can be tweaked as necessary to produce a high-quality draft assembly. For the LACHESIS publication, these parameters were set to values that are appropriate for the datasets that went into those assemblies, and these are the values you can see in the INI files that are provided with the LACHESIS distribution. However, these values may not work for your situation. The ideal values depend on the repeat content of the genome, the N50 of your input assembly, your density of Hi-C data, and your preference for accuracy versus completeness.

Try tweaking some of the parameters and re-running LACHESIS. The easiest approach is to produce a good clustering result first, then go from there to ordering (and make sure to set OVERWRITE_CLMS = 1 when you move from clustering to ordering.) If you have a reference genome to compare your result against (USE_REFERENCE = ), the reference-based evaluation will be very helpful.

COPYRIGHT AND DISCLAIMER

The LACHESIS software package and all software and documentation contained with it are copyright © 2012-2013 by Josh Burton and the University of Washington. All rights are reserved.

This software is supplied 'as is' without any warranty or guarantee of support. The University of Washington is not responsible for its use, misuse, or functionality. In no event shall the authors or copyright holders be liable for any claim, damages, or other liability arising from, out of, or in connection with this software.

ACKNOWLEDGEMENTS

Thanks to Jay Shendure for leadership, management, and many ideas.

Thanks to Aaron McKenna, Qi Zhou, and Christopher Beitel for patiently helping me test LACHESIS for bugs and compatibility.

Thanks to Aaron McKenna for helping make LACHESIS available over GitHub.