1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
|
ABySS - assemble short reads into contigs
* Compiling ABySS
Compiling ABySS should be as easy as
./configure && make
To install ABySS in a specified directory
./configure --prefix=/opt/ABySS && make && sudo make install
If you wish to build the parallel assembler with MPI support,
MPI should be found in /usr/include and /usr/lib or its location
specified to configure:
./configure --with-mpi=/usr/lib/openmpi && make
ABySS should be built using Google sparsehash to reduce memory usage,
although it will build without. Google sparsehash should be found in
/usr/include or its location specified to configure:
./configure CPPFLAGS=-I/usr/local/include
The default maximum k-mer size is 64 and may be decreased to reduce
memory usage or increased at compile time:
./configure --enable-maxk=96 && make
To run ABySS, its executables should be found in your PATH.
* Single-end assembly
Assemble short reads in a file named reads.fa into contigs in a
file named contigs.fa with the following command:
ABYSS -k25 reads.fa -o contigs.fa
where -k is an appropriate k-mer length. The only method to find the
optimal value of k is to run multiple trials and inspect the results.
The following shell snippet will assemble for every value of k from 20
to 40.
for k in {20..40}; do
ABYSS -k$k reads.fa -o contigs-k$k.fa
done
The maximum value for k is 64. This limit may be changed at compile
time using the --enable-maxk option of configure. It may be decreased
to 32 to decrease memory usage, which is particularly useful for large
parallel jobs, or increased to 96.
* Paired-end assembly
To assemble paired short reads in two files named reads1.fa and
reads2.fa into contigs in a file named ecoli-contigs.fa, run the
command:
abyss-pe k=25 n=10 in='reads1.fa reads2.fa' name=ecoli
where k is the k-mer length as before.
n is the minimum number of pairs needed to consider joining two
contigs. The optimal value for n must be found by trial.
in specifies the input files to read, which may be in FASTA, FASTQ,
qseq, export, SAM or BAM format and compressed with gz, bz2 or xz and
may be tarred.
The assembled contigs will be stored in ${name}-contigs.fa.
The suffix of the read identifier for a pair of reads must be one of
'1' and '2', or 'A' and 'B', or 'F' and 'R', or 'F3' and 'R3', or
'forward' and 'reverse'. The reads may be interleaved in the same file
or found in different files. If the mates are in different files, it's
highly recommended to place each pair of files adjacent on the command
line and to use an even number of threads. Even if you are running on
a single-processor machine, using two threads will help performance.
Do not group together all the files containing the forwards reads
followed by all the files containing the reverse reads.
Reads without mates should be placed in a file specified by the `se'
(single-end) parameter. Reads without mates in the paired-end files
will slow down the paired-end assembler considerably during the
ParseAligns stage.
abyss-pe is a driver script implemented as a Makefile and runs a
single-end assembly, as described above, and the following commands,
which must be found in your PATH:
ABYSS - the single-end assembler
AdjList - finds overlaps of length k-1 between contigs
PopBubbles - collapses variation
KAligner - aligns reads to contigs
ParseAligns - finds pairs of reads in alignments
DistanceEst - estimates distances between contigs
Overlap - find overlaps between blunt contigs
SimpleGraph - finds paths between pairs of contigs
MergePaths - merges consistent paths
Consensus - for a colour-space assembly, convert the colour-space
contigs to nucleotide contigs
* Paired-end assembly of multiple fragment libraries
The distribution of fragment sizes of each library is calculated
empirically by aligning paired reads to the contigs produced by the
single-end assembler, and the distribution is stored in a file with
the extension .hist, such as ecoli-4.hist. The N50 of the single-end
assembly must be well over the fragment-size to obtain an accurate
empirical distribution.
Here's an example scenario of assembling a data set with two different
fragment libraries and single-end reads:
Library lib1 has reads in two files, lib1_1.fa and lib1_2.fa.
Library lib2 has reads in two files, lib2_1.fa and lib2_2.fa.
Single-end reads are stored in two files se1.fa and se2.fa.
The command line to assemble this example data set is...
abyss-pe k=25 n=10 name=ecoli lib='lib1 lib2' \
lib1='lib1_1.fa lib1_2.fa' lib2='lib2_1.fa lib2_2.fa' \
se='se1.fa se2.fa'
The empirical distribution of fragment sizes will be stored in two
files named lib1-3.hist and lib2-3.hist. These files may be plotted to
check that the empirical distribution agrees with the expected
distribution. The assembled contigs will be stored in
${name}-contigs.fa.
* Parallel assembly
The `np' option of abyss-pe specifies the number of processes to
use for the ABYSS-P parallel MPI job. Without any MPI configuration,
this will allow you to make use of multiple cores on a single machine.
To use multiple machines for assembly, you must create a hostfile for
mpirun, which is describe in the mpirun man page.
The paired-end assembly stage is multithreaded but runs on a single
machine. The number of threads to use may be specified with the
parameter j. The default is the same as np. ABySS is multithreaded
using pthread and OpenMP, which requires a modern compiler such as
GCC 4.3 or greater.
Open MPI integrates well with SGE (Sun Grid Engine). For example, to
submit an array of jobs to assemble every odd value of k between 51
and 63 using 64 processes for each job:
qsub -pe openmpi 64 -t 51-63:2 -N testing abyss-pe in=reads.fa n=10
For more information on using SGE and qsub, please refer to the qsub
manual page. Open MPI must have been compiled with support for SGE
using the ./configure --with-sge option.
* See also
Try `ABYSS --help' for more information on command line options, or
see the manual page in the files `ABYSS.1' and `abyss-pe.1'.
Please refer to the mpirun manual page for information on configuring
parallel jobs.
Written by Jared Simpson and Shaun Jackman.
Subscribe to the users' mailing list at
http://groups.google.com/group/abyss-users
Contact the users' mailing list at <abyss-users@googlegroups.com>
or the authors directly at <abyss@bcgsc.ca>.
|