NaS is a hybrid approach developed to take advantage of data generated using MinION device. We combine Illumina and Oxford Nanopore technologies to produce NaS (Nanopore Synthetic-long) reads of up to 60 kb that aligned with no error to the reference genome and spanned repetitive regions.
NaS uses the MinION® read as a template to recruit Illumina reads and, by performing a local assembly, build a high-quality synthetic read. In the first step, a stringent alignment using BLAT (fast mode) or LAST (sensitive mode) is performed to retrieve Illumina short reads and their complementary sequences, called seed-reads. Next, the seed-read set is extended by searching for similar reads and their complementary sequences in the initial set using Compareads (two reads are considered similar if they share several common k-mers). This second step is crucial to retrieve Illumina reads that correspond to low-quality regions of the template. Finally, a microassembly of the reads is performed, instead of a classical polishing of the consensus, using an overlap-layout-consensus strategy, and repeats are resolved by a graph traversal algorithm.
At the moment NaS should only be used on genomes smaller than 30 Mb, to avoid long running times.
NaS is distributed open-source under CeCILL FREE SOFTWARE LICENSE. Check out http://www.cecill.info/ for more information about the contents of this license.
NaS home on the web is http://www.genoscope.cns.fr/nas
NaS is also available on Docker Hub http://registry.hub.docker.com/u/rdbioseq/nas/
Contact : nas [a] genoscope [.] cns [.] fr
- A Linux based operating system.
- Binaries are provided for the following platform : Linux x86_64 (but some version like Ubuntu may not be compatible, see Troubleshooting)
- Shell tool GNU Parallel ( http://www.gnu.org/software/parallel/ ) at least (22052015)
- Perl 5.8.0 or higher installed.
- Perl graph library (http://search.cpan.org/~jhi/Graph/)
- Perl GetOpt module (http://search.cpan.org/dist/Getopt-Long/)
- Perl Set::IntSpan module (http://search.cpan.org/~swmcd/Set-IntSpan-1.19/IntSpan.pm)
- Newbler assembler v2.9 (available from 454 website)
- Blat binary (at least v35) accessible through your PATH environnment variable
- Last binary (at least 588) accessible through your PATH environnment variable
The two following binaries : fastalength and fastacomposition come from the exonerate software availaible under the LGPL license. See http://www.ebi.ac.uk/~guy/exonerate for details. The two following binaries : compareads2 and extrac_reads come from the Compareads software available under the GPL license. See http://colibread.inria.fr/software/compareads/ for details.
- Clone this GitHub repository
- Modify path to newbler and blat binaries (in NaS script, your PATH variable)
- Modify if needed the Perl and sh interpreters that have been set to : /usr/bin/perl and /bin/sh
- To test the program download this example dataset
wget http://www.genoscope.cns.fr/externe/nas/datasets/NaS_example_acineto.tar.gz - Untar/unzip the archive :
tar -zxvf NaS_example_acineto.tar.gz - Run NaS :
$(pwd)/NaS_v2/NaS --fq1 NaS_example_acineto/AWK_DOSF_1_1_A5KR6.IND3_clean.10prc.fastq --fq2 NaS_example_acineto/AWK_DOSF_1_2_A5KR6.IND3_clean.10prc.fastq --nano NaS_example_acineto/MinION_reads_Acinetobacter_baylyi.fa --out NaS_example --mode sensitive --nb_proc 5
This part describes the different steps required to run NaS
The directory "NaS_example_acineto" provides an example of a NaS run performed on 5 MinION reads from Acinetobacter baylyi ADP1.
- MinION reads : NaS_example_acineto/MinION_reads_Acinetobacter_baylyi.fa
Sequence name line should not contain slash
- Illumina reads : NaS_example_acineto/AWK_DOSF_1_?_A5KR6.IND3_clean.10prc.fastq
Warning : At the moment, we only support the following fastq format :
- The sequence name line have to be a one field line
- Sequence names have to end with '/1' (read1) and '/2' (read2)
Example :
@M2:A5KR6:1:1101:9590:1008/1
ACTCAAAGAACAAGAGTTACAGTCTAAAAAAGCTGCGGTTGC...
+
8ACCGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGG...
#################################################
#################################################
--fq1 : illumina reads (R1) in fastQ format
--fq2 : illumina reads (R2) in fastQ format
--nano : nanopore long reads in fastA format
--out : Output directory, default is NaS_date_pid
--mode : mode : fast or sensitive. fast mode use blat as aligner whereas sensitive mode use last, default is fast
--tile : ONLY in fast mode : tile size parameter of blat, default is 10
--step : ONLY in fast mode : step size parameter of blat, default is 5
--a : ONLY in sensitive mode : gap existence cost parameter of last, default is 1
--b : ONLY in sensitive mode : gap extension cost parameter of last, default is 1
--e : ONLY in sensitive mode : score threshold parameter of last, default is 40
--t : number of substrings for compareads, default is 3
--k : size of substrings for compareads, default is 32
--nb_proc : Number of parallel task, default is 1
-h : help message
$(pwd)/NaS_v2/NaS --fq1 NaS_example_acineto/AWK_DOSF_1_1_A5KR6.IND3_clean.10prc.fastq --fq2 NaS_example_acineto/AWK_DOSF_1_2_A5KR6.IND3_clean.10prc.fastq --nano NaS_example_acineto/MinION_reads_Acinetobacter_baylyi.fa --out NaS_example --mode sensitive --nb_proc 5
Command : /env/cns/src/NaS/NaS_v5.0_evolution/NaS_wrapped --fq1 NaS_example_acineto/AWK_DOSF_1_1_A5KR6.IND3_clean.10prc.fastq --fq2 NaS_example_acineto/AWK_DOSF_1_2_A5KR6.IND3_clean.10prc.fastq --nano NaS_example_acineto/MinION_reads_Acinetobacter_baylyi.fa --out NaS_example --mode sensitive --nb_proc 5
[jeu. août 13 15:46:24 CEST 2015] Create output directory : NaS_example
[jeu. août 13 15:46:24 CEST 2015] Create fasta file from fastq...
[jeu. août 13 15:47:22 CEST 2015] Alignement step in sensitive mode...
[jeu. août 13 15:48:37 CEST 2015] Convert maf file to psl file...
[jeu. août 13 15:48:48 CEST 2015] Select reads...
[jeu. août 13 15:48:48 CEST 2015] Retrieve similar reads...
[jeu. août 13 15:51:13 CEST 2015] Generate NaS reads...
[jeu. août 13 15:51:26 CEST 2015] Untangle complex NaS reads...
[jeu. août 13 15:51:27 CEST 2015] Generate statistics...
NbReads= 5 CumulativeSize= 31008 N50size= 7994 minSize= 2512 maxSize= 10464 avgSize= 6201.6 => NaS_example/NANO_reads.stats
NbReads= 5 CumulativeSize= 37517 N50size= 9743 minSize= 1982 maxSize= 12787 avgSize= 7503.4 => NaS_example/NaS_hqctg_reads.stats
[jeu. août 13 15:51:27 CEST 2015] Delete temporary file...
[jeu. août 13 15:51:30 CEST 2015] Total execution time with 5 core(s) : [00:05:06]
fastalength: error while loading shared libraries: libglib-1.2.so.0: cannot open shared object file: No such file or directory
fastacomposition: error while loading shared libraries: libglib-1.2.so.0: cannot open shared object file: No such file or directory
This error message appears when exonerate binaries (fastalength and fastacompisition) provided with NaS are not compatible with your OS. To fix this issue, download sources from exonerate website: http://www.ebi.ac.uk/~guy/exonerate/ and compile them. Replace files : fastalength and fastacompisition in NaS_v2 directory.
Unknown option: cat
Unknown option: cat
This error message appears when your version of parallel is too old. Update parallel to at least parallel22052015
The size of kmers selected must be between 21 and 38 (problems occurs for a smaller or greater size)
This work was financially supported by the Genoscope, Institut de Genomique, CEA and Agence Nationale de la Recherche (ANR), and France Génomique (ANR-10-INBS-09-08).
Jean-Marc Aury, Amin Madoui, Stefan Engelen, Guillaume Gautreau, François-Xavier Babin and Benjamin Istace