gargammel is a set of programs aimed at simulating ancient DNA fragments. For ancient hominin samples our program can also simulate various levels of present-day human contamination and microbial contamination.
The website for gargammel can be found here: https://grenaud.github.io/gargammel/
If you have Github account, consider creating an issue, you will help others who might have the same problem.
contact: Gabriel Renaud
email: gabriel [dot] reno [ at sign ] gmail.com
I accept pull request for novel features.
Do a :
git clone --recursive --depth 1 https://github.com/grenaud/gargammel.git
or via (bio)conda
conda install -c bioconda gargammelInstalling with conda will only provide the main gargammel program, for the additional scripts in the repository, please run
git cloneas above, and create the conda environment described below.
- git
- C++ compiler supporting C++11
- cmake, you can install on Ubuntu by typing: sudo apt install cmake
- zlib
- lib gsl, you can install on Ubuntu by typing: sudo apt-get install libgsl0-dev
If you plan on using ms2chromosomes.py to simulate chromosomes based on ms, you also need:
- Hudson's ms (see: http://home.uchicago.edu/rhudson1/source/mksamples.html)
- seq-gen, you can install on Ubuntu by typing: sudo apt install seq-gen
Both should be installed in your path.
Alternatively, you can use the supplied conda environment.yml file to download and set up all dependencies described in this README for you.
conda env create -f environment.yml
If you are using the conda enviroment, you can skip this step and just load the environment with
conda activate gargammel. All subsequent steps you can replacegargammel.plwith justgargammel.
In the main directory, simply type
make
This should install bamtools (C++ library to read/write BAM files) and ART (Illumina read simulator).
The main driver script, gargammel.pl calls the following programs in order to simulate the in vivo process by which ancient DNA fragments are retrieved:
- fragSim: simulation of ancient DNA fragments being retrieved at random from the genome
- deamSim: simulation of damage to the fragments selected by fragSim
- adptSim: adding of adapters to create raw Illumina reads (without errors and quality scores)
Finally, the simulated raw Illumina reads are sent to ART to add sequencing errors and corresponding quality scores.
The basic input is a directory with 3 subfolders named:
- endo/
- cont/
- bact/
Which represent the endogenous ancient human, the present-day human contaminant and the microbial contamination respectively. Each file inside represents a genome (not simply a chromosome or scaffold). The endogenous ancient human can only contain more than 2 genomes since it is a diploid individual. For the microbial contamination, please add a representative set of microbes for your sample (see the section about the examples of microbial databases).
This is an example of usage to simulate a slightly contaminated (8%) dataset. First, we will simulate chromosomes using ms and seq-gen:
mkdir data
Next, we will create 1000 simulations of 2 lineages that are allowed to coalesce after 0.2 units of coalescence. The first one will represent our endogenous ancient human while the other, the present-day human contaminant. It will also generate an additional chromosome from the same population as the contaminant to be used as reference for alignment. We generate sequences for those using the following script:
cd data/
python ../ms2chromosomes.py -s 0.2 -f . -n 1000
rm -rfv simul_* seedms #cleanup
This will create the following files:
cont/cont.0.fa
cont/cont.1.fa
endo/endo.1.fa
endo/endo.2.fa
endo/segsites
ref.fa
The segsites files correspond to heterozygous sites between both endogenous genomes.
Then we will create the aDNA fragments:
cd ..
./gargammel.pl -c 3 --comp 0,0.08,0.92 -f src/sizefreq.size.gz -matfile src/matrices/single- -o data/simulation data/
This will simulate a dataset with 8% human contamination. The rate of misincorporation due to deamination that will be used will follow a single-strand deamination using the empirical rates measured from the Loschbour individual from:
Lazaridis, Iosif, et al. "Ancient human genomes suggest three ancestral populations for present-day Europeans." Nature 513.7518 (2014): 409-413.
The size distribution of the aDNA fragments is a subset of:
Fu, Qiaomei, et al. "Genome sequence of a 45,000-year-old modern human from western Siberia." Nature 514.7523 (2014): 445-449.
The read size will be 2x75bp and the Illumina platform being simulated is the HiSeq 2500. The final reads will be found:
data/out_s1.fq.gz
data/out_s2.fq.gz
Here are further examples of usage:
- Low coverage 0.5X coverage with fragments of length 40:
gargammel.pl -c 0.5 --comp 0,0,1 -l 40 -o data/simulation data/
- Generating exactly 1M fragments of length with a log-normal distribution of location 4.106487474 and scale 0.358874723:
gargammel.pl -n 1000000 --comp 0,0,1 --loc 4.106487474 --scale 0.358874723 -o data/simulation data/
- High coverage (20X) with high amount of present-day contamination (40%) with fragments of length 45:
gargammel.pl -c 20 --comp 0,0.4,0.6 -l 45 -o data/simulation data/
- Evaluating the impact of mapping 1M fragments with length 40 without double-stranded deamination:
gargammel.pl -n 1000000 --comp 0,0,1 -l 40 -o data/simulation data/
- Evaluating the impact of mapping 1M fragments with length 40 with double-stranded deamination:
gargammel.pl -n 1000000 --comp 0,0,1 -l 40 -damage 0.03,0.4,0.01,0.3 -o data/simulation data/
- Generate a single-end run of 96 cycles on a HiSeq 2500 Illumina run with 1M fragments of 40bp:
gargammel.pl -n 1000000 --comp 0,0,1 -l 40 -rl 96 -se -ss HS25 -o data/simulation data/
- Generate a paired-end run of 96 cycles on a HiSeq 2500 Illumina run with 1M fragments of 40bp:
gargammel.pl -n 1000000 --comp 0,0,1 -l 40 -rl 96 -ss HS25 -o data/simulation data/
If you use gargammel.pl or deamSim, you can speficiy deamination/damage using either:
-
Use Briggs model parametes (see Briggs, Adrian W., et al. "Patterns of damage in genomic DNA sequences from a Neandertal." Proceedings of the National Academy of Sciences 104.37 (2007): 14616-14621.)
-
Use a misincorporation matrix computed by mapDamage (https://ginolhac.github.io/mapDamage). This matrix is in the results directory created by mapDamage and is called "misincorporation.txt". There are 2 examples of such files:
examplesMapDamage/results_LaBrana/misincorporation.txt examplesMapDamage/results_Ust_Ishim/misincorporation.txt
The first is from a double-stranded library and the second a single-stranded one. To use either, you can use the wrapper script or deamSim as such:
-mapdamage examplesMapDamage/results_LaBrana/misincorporation.txt double
-mapdamage examplesMapDamage/results_Ust_Ishim/misincorporation.txt single
We suggest that you run mapDamage on the empirical data that you are trying to emulate and use the resulting misincorporation.txt file.
-
Specify a matrix of deamination rates, we use the following format, the first line is the header:
A->C A->G A->T C->A C->G C->T G->A G->C G->T T->A T->C T->G pos rate_{A->C} rate_{A->G} rate_{A->T} rate_{C->A} rate_{C->G} rate_{C->T} rate_{G->A} rate_{G->C} rate_{G->T} rate_{T->A} rate_{T->C} rate_{T->G}
The pos. is the position 0,1... after the fragment beginning/end. The rate is specified using the following: estimate [estimate_low estimate_high]. For example, 0.3 [0.2 0.4] means that the rate of deamination is 0.3 or 30%.
example of a format:
A->C A->G A->T C->A C->G C->T G->A G->C G->T T->A T->C T->G
0 1.853e-3 [1.726e-3..1.989e-3] 4.064e-3 [3.875e-3..4.263e-3] 3.269e-3 [3.099e-3..3.448e-3] 6.661e-3 [6.254e-3..7.094e-3] 3.057e-3 [2.785e-3..3.355e-3] 8.004e-2 [7.865e-2..8.145e-2] 1.236e-2 [ 1.183e-2..1.292e-2] 4.131e-3 [3.828e-3..4.459e-3] 6.703e-3 [6.314e-3..7.116e-3] 3.845e-3 [3.624e-3..4.079e-3] 4.581e-3 [4.339e-3..4.836e-3] 2.169e-3 [2.005e-3..2.347e-3]
1 1.986e-3 [1.849e-3..2.134e-3] 4.273e-3 [4.070e-3..4.487e-3] 3.030e-3 [2.859e-3..3.211e-3] 5.357e-3 [5.001e-3..5.738e-3] 3.188e-3 [2.916e-3..3.485e-3] 1.427e-2 [1.369e-2..1.488e-2] 9.514e-3 [ 9.075e-3..9.974e-3] 3.316e-3 [3.061e-3..3.593e-3] 5.061e-3 [4.743e-3..5.400e-3] 3.421e-3 [3.216e-3..3.639e-3] 4.865e-3 [4.620e-3..5.124e-3] 2.201e-3 [2.038e-3..2.377e-3]
This follows the output of https://bitbucket.org/ustenzel/damage-patterns.git
-
You can use one of the precalculated rates of deamination in src/matrices/. There is a damage from single-strand and a double-strand libraries from the following study:
Lazaridis, Iosif, et al. "Ancient human genomes suggest three ancestral populations for present-day Europeans." Nature 513.7518 (2014): 409-413.
See the methylation question for adding different rates of deamination for methylated/unmethylated cytosine.
Can I specify different rates of misincorporation due to deamination for the endogenous/bacterial/human contaminant sources?
Yes, please refer to the options of the wrapper script gargammel.pl
Yes. In the endogenous genome, specify methylated cytosine as 'c' (lowercase c) and unmethylated cytosine as 'C' (uppercase C). You can specify multiple cells using the following directory structure:
input/
input/endo
input/endo/C0
input/endo/C0/chr20_0_split1.fa
input/endo/C0/chr20_0_split1.fa.fai
input/endo/C0/chr20_0_split2.fa
input/endo/C0/chr20_0_split2.fa.fai
input/endo/C1
input/endo/C1/chr20_1_split1.fa
input/endo/C1/chr20_1_split1.fa.fai
input/endo/C1/chr20_1_split2.fa
input/endo/C1/chr20_1_split2.fa.fai
input/endo/C2
input/endo/C2/chr20_2_split1.fa
input/endo/C2/chr20_2_split1.fa.fai
input/endo/C2/chr20_2_split2.fa
input/endo/C2/chr20_2_split2.fa.fai
Where C0 reprensents the first cell, C1 the second and so forth. A lower case C 'c' is a methylated C and an uppercase 'C' is a an unmethylated C. To create these files from a reference and a methylation map, please see the script src/addMethyl.pl which needs to be modified (hardcoded paths).
Methylated and unmethylated cytosines on the - strand can be specified using 'g' and 'G'. Once this is done, you can specify the option: --methyl for gargammel.pl. When using --methyl, you can then specify different matrix files for rates of deamination for nonmethylated and methylated cytosines:
-matfilenonmeth [matrix file prefix] Read the matrix file of substitutions for non-methylated Cs
-matfilemeth [matrix file prefix] Read the matrix file of substitutions for methylated Cs
By composition we mean the base frequency at the breaks. You could generate it manually, the format is as follows:
# comment
Chr End Std Pos A C G T Total
[chr] ['5p' or '3p'] ['+' or '-'] [pos wrt the 5p/3p end] [count A] [count C] [count G] [count T] [sum of counts]
For instance:
# table produced by mapDamage version 2.0.5-1-ge06bd84
# using mapped file Ust_Ishim.hg19_1000g.bam and human_g1k_v37.fasta as reference file
# Chr: reference from sam/bam header, End: from which termini of DNA sequences, Std: strand of reads
Chr End Std Pos A C G T Total
21 3p + -4 177086 83624 114115 150943 525768
21 3p + -3 191241 80099 104155 150269 525764
21 3p + -2 197747 63995 127660 136360 525762
21 3p + -1 180637 49770 79519 215833 525759
21 3p + 1 188505 79678 204246 53417 525846
21 3p + 2 156848 74009 128222 166767 525846
21 3p + 3 188608 75382 113613 148243 525846
21 3p + 4 173245 84205 117226 151170 525846
The lines above specify the base count close +/- 4 bases to the 3p end for fragments mapping to the + strand. An example of this type of file is found here: src/dnacomp.txt
Such a file can be generated using mapDamage2.0:
Jonsson, Hakon, et al. "mapDamage2.0: fast approximate Bayesian estimates of ancient DNA damage parameters." Bioinformatics (2013): btt193.
It is normally called "dnacomp.txt" in the output directory, you can filter a single chromosome (in this case 21) using this command:
grep "^21\|^#\|^Chr" /path to mapDamage output/results_[sample name]/dnacomp.txt > dnacomp.txt
Ancient DNA molecules tend to be fragmented and can be very short but tend to have a specific shape. Both for the wrapper script (gargammel.pl) and the fragment simulation program (fragSim), there are are 4 ways to specify the :
- Specify a fixed length using -l
- Open a file containing the size distribution using -s, one empirical fragment length per line eg:
82
95
66
144
87
68
74
48
77
43
- Open a file containing the size frequencies using -f in the format "size[TAB]freq" eg:
40 0.017096
41 0.01832
42 0.0201954
43 0.018399
44 0.0195637
45 0.0198993
46 0.0196822
47 0.0209456
48 0.0203929
49 0.0199783
50 0.0204323
- Specify the size distribution using parameters from a log-normal distribution, using options --loc and --scale.
If you wish to specify the aDNA fragment size distribution as a log-normal, you can use the following script to infer the location and scale parameters:
#!/usr/bin/env Rscript-3.2.0
library(fitdistrplus)
library(MASS)
args=(commandArgs(TRUE))
data <- read.table(args[1]);
df<-fitdistr(data$V1, "lognormal")
print(df);
You can change the header to suit the version of R that you have.
For the input/bact/ directory which represent the microbial contamination, gargammel needs a set of fasta files that represent the different microbes. Each file corresponds to exactly one microbial species. Each fasta file must contain the genome of the microbial species, multiple scaffolds and plasmids are allowed. Each fasta file must also be faidx indexed. This directory must also contain a file called "list". This file contains the list of every fasta files in that directory along with their relative abundance in the desired bacterial contamination. For example:
bacteria1.fa 0.5
bacteria2.fa 0.3
bacteria3.fa 0.2
The abundance will be printed on the console when the program is launched. Some users have reported discrepancies between the original bacterial abundance and the printed one. Make sure that they are equal and that the bacterial abundance file uses UNIX carriage returns (use dos2unix or mac2unix to transform from DOS/MAC to Unix format).
If you wish to download an example of a suitable bacterial database, you can simply type:
make bacterialex
this will create a directory called bactDBexample/ which contains clovis/ and k14/, the profiled microbial communities from Rasmussen et al. "The genome of a Late Pleistocene human from a Clovis burial site in western Montana." Nature 506.7487 (2014): 225-229. and Seguin-Orlando et al. "Genomic structure in Europeans dating back at least 36,200 years." Science 346.6213 (2014): 1113-1118, respectively.
You can copy the files from the fasta/ directory into the input's bact/ directory as such
cp -v bacterialex/clovis/fasta/* [path to input]/bact/
metaBIT [https://bitbucket.org/Glouvel/metabit] is a metagenomic profiler from high-throughput sequencing shotgun data. To download the fasta files based on a profile obtained using metaBIT's output, simply supply the "all_taxa.tsv" file, which details the different species and their abundances, make sure you are connected to the internet and use the retrieveFromMetabit script in as such:
mkdir exampleBacteriaDB
cd exampleBacteriaDB
[copy the all_taxa.tsv in the current directory]
src/microbial_fetcher/retrieveFromMetabit all_taxa.tsv
If you wish, you can enter your email for the ftp from NCBI (to avoid getting banned from the FTP):
src/microbial_fetcher/retrieveFromMetabit all_taxa.tsv [email protected]
This will download the necessary files from NCBI to create a database suitable for gargammel to simulate microbial species in the exampleBacteriaDB/fasta and run samtools faidx on each file. You need standard UNIX utilities such as awk/sed/python/curl/wget/gzip to be installed as well as samtools. Please move the fasta/ directory produced (exampleBacteriaDB/fasta in the example above) to the input/bact/. The file named "exampleBacteriaDB/fastafasta/list" is the list of bacterial species along with their abundance. Another file, "exampleBacteriaDB/Microbial_ID.log" details the strain/ID and ftp link used. retrieveFromMetabit uses GNU parallel (see O. Tange (2011): GNU Parallel - The Command-Line Power Tool, ;login: The USENIX Magazine, February 2011:42-47.), please make sure that it is installed.
If you want to use a uniform probability instead of a weighted list, go to "input/bact" and type (if fasta files end with .fa):
total=`ls -1 input/bact/*fa |wc -l ` && ls -1 input/bact/*fa | awk -v total="$total" ' {print $1"\t"(1/total)}' > input/bact/list
metaBIT ref: Louvel et al. "metaBIT, an integrative and automated metagenomic pipeline for analyzing microbial profiles from high-throughput sequencing shotgun data." Molecular ecology resources (2016).
To provide an example of using empirical VCF files to create sequences for the simulation, there is a folder exampleSeq/ with a Makefile. This makefile provides a simple example of creating 2 chromosomes (2 endogenous sequences + 2 contaminant sequences for a diploid genome) from VCF files. This makefile needs the following commands to be installed in the path:
- bedtools
- bgzip
- tabix
- samtools
- bcftools, must support "consensus" command
Make sure that you are connected to the internet and type:
cd exampleSeq/
make
This will download the VCF files from the Altai Neanderthal (endogenous) and a present-day human of European descent (contaminant) create 4 files:
inputfolder/endo/endo.2.fa
inputfolder/endo/endo.1.fa
inputfolder/cont/cont.1.fa
inputfolder/cont/cont.2.fa
along with their respective fasta index. If you wish to add bacterial sequences to the mix, please see the section above about "Examples of bacterial databases" and you can copy some files to cont/ directory: cp -v ../bactDBexample/k14/fasta/* inputfolder/bact/
To create a sample with say 10% present-day human contamination with fragment length of 40bp, run:
./gargammel.pl -c 0.5 --comp 0,0.1,0.9 -l 40 -o exampleSeq/simulationc10 exampleSeq/inputfolder/
If you have some microbial sequences, to create a sample with say 70% bacterial content, 5% present-day human contamination and 25% endogenous, run:
./gargammel.pl -c 0.5 --comp 0.7,0.05,0.25 -l 40 -o exampleSeq/simulationb70c5 exampleSeq/inputfolder/
-
I am getting:
./art_illumina: error while loading shared libraries: libgsl.so.0: cannot open shared object file: No such file or directory
Make sure you have libgsl installed and create a symbolic link:
sudo ln -s /usr/lib/x86_64-linux-gnu/libgsl.so.23.0.0 /usr/lib/libgsl.so.0
-
Can I know where on the genome of origin the fragment was sampled?
Yes! fragSim, which generates the original fragments uses the following format: [CHROMOSOME NAME]:[STRAND]:[START]:[END]:[LENGTH] The overall wrapper (gargammel.pl) will add e1_ to endogenous fragments from the first reference and e2_ to endogenous fragments from the second reference. It will add c_X to the fragments from present-day human contaminants where X is the # of the genome and b_X to the fragments from bacterial contaminants where X is the # of the genome.
-
Can gargammel simulate indels?
Yes and no. gargammel does not currently insert indels as a result of sequencing errors. However, if you add indels in your input genome, it will handle them without any problems.
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