vijender-singh / refguideddenovoassembly_pipelines

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Reference-guided de novo assembly

by Heidi Lischer ([e-mail]), 2015/15

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We adapted and extended the reference-guided assembly approach from Schneeberger et al. [1]. The main idea of this approach is to first map reads against a reference genome of a related species to reduce the complexity of de novo assembly within continuous covered regions. In a further step, reads with no similarity to the related genome are integrated.

Reference-guided de novo assembly pipeline:

1. Step: quality/adapter trimming and quality check
2. Step: map reads against reference and define blocks and superblocks
3. Step: do deNovo assembly within superblocks
4. Step: get non-redundant supercontigs
5. Step: map reads on supercontigs and de novo assemble unmapped reads
6. Step: map reads to all supercontics and correct them
7. Step: scaffolding and gap closing

[1] http://www.pnas.org/content/early/2011/06/01/1107739108.full.pdf+html?with-ds=yes

Dependencies

(in brackets: used version of softwares)

Third party programs:

• fastqc (v 0.10.1): Fastq quality checking
• trimmomatic-0.32 (v 0.32): quality and adapter trimming
• samtools (v 1.3): Tools for alignments in the SAM format
• bcftools (v 1.3): Tools for variant calling and manipulating VCFs and BCFs
• bamtools (v 2.3.0): Tools for alignments in the BAM format
• bedtools (v 2.19.0): Tool for genomic arithmetics
• picardtools (v 1.109): Processing alignment files
• bowtie2 (v 2.2.1): NGS alignment tool
• seqtk (v 1.0-r45): Fast Fasta/Fastq manipulation tool
• AMOScmp (v 3.1.0): Comprarative genome assembly
• MUMmer (v 3.23): Comprarative genome assembly
• GenomeAnalysisTK (v 3.1-1): Genome analysis toolkit
• SOAPdenovo2 (v r240): De novo genome assembler and scaffolder

and one of these de novo assemblers:

• AllPaths-LG (v 51279)
• idba (v 1.1.1)
• abyss-pe (v 1.5.2)
• SOAPdenovo2 (v r240)

Additional in-house scripts

• RemoveShortSeq.jar: Remove short sequences and make unique identifiers from FASTA/FASTQ
• GetBlocks_new.jar: Create blocks and superblocks
• FastaToAmos.jar: Transform FASTA to Amos format
• WriteSoapConfig.jar: writes a SOAP config file
• FastaStats.jar: outputs FASTA statistics
• SplitSeqLowCov.jar: splits sequences at low coverage

Running

In first lines of the main scripts you have to adapt the paths to your system. (Everything between:

# set variables #########################################
 ...
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If the parameters are set you can run the pipeline as follows: bash refGuidedDeNovoAssembly_ALLPATHS.sh or bash refGuidedDeNovoAssembly_IDBA.sh or bash refGuidedDeNovoAssembly_ABYSS.sh or bash refGuidedDeNovoAssembly_SOAP.sh