BOL: Related items

miniasm: very fast OLC-based de novo assembler for noisy long reads

Jit — Mon, 27 Nov 2017 07:58:49 -0600

Miniasm is a very fast OLC-based de novo assembler for noisy long reads. It takes all-vs-all read self-mappings (typically by minimap) as input and outputs an assembly graph in the GFA format. Different from mainstream assemblers, miniasm does not have a consensus step. It simply concatenates pieces of read sequences to generate the final unitig sequences. Thus the per-base error rate is similar to the raw input reads.

So far miniasm is in early development stage. It has only been tested on a dozen of PacBio and Oxford Nanopore (ONT) bacterial data sets. Including the mapping step, it takes about 3 minutes to assemble a bacterial genome. Under the default setting, miniasm assembles 9 out of 12 PacBio datasets and 3 out of 4 ONT datasets into a single contig. The 12 PacBio data sets are PacBio E. coli sample, ERS473430, ERS544009, ERS554120, ERS605484, ERS617393, ERS646601, ERS659581, ERS670327, ERS685285, ERS743109 and a deprecated PacBio E. coli data set. ONT data are acquired from the Loman Lab.

For a C. elegans PacBio data set (only 40X are used, not the whole dataset), miniasm finishes the assembly, including reads overlapping, in ~10 minutes with 16 CPUs. The total assembly size is 105Mb; the N50 is 1.94Mb. In comparison, the HGAP3produces a 104Mb assembly with N50 1.61Mb. This dotter plot gives a global view of the miniasm assembly (on the X axis) and the HGAP3 assembly (on Y). They are broadly comparable. Of course, the HGAP3 consensus sequences are much more accurate. In addition, on the whole data set (assembled in ~30 min), the miniasm N50 is reduced to 1.79Mb. Miniasm still needs improvements.

Miniasm confirms that at least for high-coverage bacterial genomes, it is possible to generate long contigs from raw PacBio or ONT reads without error correction. It also shows that minimap can be used as a read overlapper, even though it is probably not as sensitive as the more sophisticated overlapers such as MHAP and DALIGNER. Coupled with long-read error correctors and consensus tools, miniasm may also be useful to produce high-quality assemblies.

Minimap and miniasm are ultrafast tools for (i) mapping and (ii) assembly. Designed for long, noisy reads, they do not have a correction or consensus step, and therefore the resulting assemblies are contiguous (i.e. long) but very noisy (i.e. full of errors)

We start with an all against all comparison:

minimap -Sw5 -L100 -m0 -t8 reads.fq reads.fq | gzip -1 > reads.paf.gz

Then we can assemble

miniasm -f reads.fq reads.paf.gz > reads.gfa

Convert GFA to FASTA:

awk '/^S/{print ">"$2"\n"$3}' reads.gfa | fold > reads.fa

And then count how many contigs:

grep ">" reads.fa | wc -l

# Download sample PacBio from the PBcR website
wget -O- http://www.cbcb.umd.edu/software/PBcR/data/selfSampleData.tar.gz | tar zxf -
ln -s selfSampleData/pacbio_filtered.fastq reads.fq
# Install minimap and miniasm (requiring gcc and zlib)
git clone https://github.com/lh3/minimap && (cd minimap && make)
git clone https://github.com/lh3/miniasm && (cd miniasm && make)
# Overlap
minimap/minimap -Sw5 -L100 -m0 -t8 reads.fq reads.fq | gzip -1 > reads.paf.gz
# Layout
miniasm/miniasm -f reads.fq reads.paf.gz > reads.gfa

Address of the bookmark: https://github.com/lh3/miniasm

Run miniasm assembler on nanopore reads !

Jit — Mon, 18 Dec 2017 04:07:50 -0600

Find the detail of the reads repeats:

fq2fa ONT_A.fastq ONT_A.fasta

minimap2 -xava-ont ONT_A.fasta ONT_A.fasta -t10 -X > AONT.paf

awk '{if($1==$6){print}}' AONT.paf > AONTself.paf

awk '$5=="-"' AONTself.paf | awk '{print $1}'| sort|uniq > invertedrepeat.list

Generated a few palindrome and repeats plots (highlighting only repeats largest than 10, 20 and 30 kb)

minidot -f 5 -m 30000 AONTself.paf > AONTself30000.eps
sed 's/_template_pass_FAH31515//' AONTself30000.eps > AONTself30000final.eps

minidot -f 5 -m 20000 AONTself.paf > AONTself20000.eps
sed 's/_template_pass_FAH31515//' AONTself20000.eps > AONTself20000final.eps

minidot -f 5 -m 10000 AONTself.paf > AONTself10000.eps
sed 's/_template_pass_FAH31515//' AONTself10000.eps > AONTself10000final.eps

Assemble with miniasm:

miniasm -f ONT_A.fasta AONT.paf > AONT.gfa
grep '^S' AONT.gfa |awk '{print ">"$2"\n"$3}' > AONT_miniasm.fasta

minimap2 -xasm10 AONT_miniasm.fasta AONT_miniasm.fasta -t1 -X > AONT_miniasm.paf

awk '{if($1==$6){print}}' AONT_miniasm.paf > AONT_miniasm_self.paf

minidot -f 5 -m 10000 AONT_miniasm_self.paf > AONT_miniasm_self10000.eps

Njoy the assembly !

Frequent Paired-end reads (PE 2x100) mapping command lines

Jit — Tue, 15 May 2018 08:59:29 -0500

bowtie2 -x hs37m -X 650 -q -1 r1.fq -2 r2.fq -S r12.bowtie2.sam

bwa aln hs37m.fa r1.fq > r1.sai && bwa aln hs37m.fa r2.fq > r2.sai \
&& bwa sampe hs37m r1.sai r2.sai r1.fq r2.fq > r12.bwa.sam

bwa bwasw ../index/bwa/hs37m.fa r12.fq > r12.bwasw.sam

gsnap -A sam -d hs37m r1.fq r2.fq > r12.gsnap.sam

novoalign -r Random -o SAM -f r1.fq r2.fq -i 500 50 -d hs37m-k14s3.novo > r12.novo.sam

smalt map -f samsoft -i 650 -o r12.smalt-k20s13.sam hs37m-k20s13 r1.fq r2.fq

stampy.py -g hs37m -h hs37m -o r12.stampy.sam -M r1.fq,r2.fq

soap -D hs37m.fa.index -a r1.fq -b r2.fq -l 32 -g 3 -u dummy -2 dummy -o r12.soap

minialign: fast and accurate alignment tool for PacBio and Nanopore long reads

Jit — Thu, 24 May 2018 08:33:26 -0500

Minialign is a little bit fast and moderately accurate nucleotide sequence alignment tool designed for PacBio and Nanopore long reads. It is built on three key algorithms, minimizer-based index of the minimap overlapper, array-based seed chaining, and SIMD-parallel Smith-Waterman-Gotoh extension.

Address of the bookmark: https://github.com/ocxtal/minialign

EAGLER: a scaffolding tool for long reads.

Jit — Mon, 04 Jun 2018 05:26:03 -0500

EAGLER is a scaffolding tool for long reads. The scaffolder takes as input a draft genome created by any NGS assembler and a set of long reads. The long reads are used to extend the contigs present in the NGS draft and possibly join overlapping contigs. EAGLER supports both PacBio and Oxford Nanopore reads.

The tool should be compatible with most UNIX flavors and has been successfully tested on the following operating systems:

Mac OS X 10.11.1
Mac OS X 10.10.3
Ubuntu 14.04 LTS

https://bib.irb.hr/datoteka/844447.Diplomski_2015_Luka_terbi.pdf

Address of the bookmark: https://github.com/mculinovic/EAGLER

Breakpointer: using local mapping artifacts to support sequence breakpoint discovery from single-end reads

Jit — Tue, 12 Jun 2018 12:41:10 -0500

Breakpointer is a fast tool for locating sequence breakpoints from the alignment of single end reads (SE) produced by next generation sequencing (NGS). It adopts a heuristic method in searching for local mapping signatures created by insertion/deletions (indels) or more complex structural variants(SVs).

Address of the bookmark: https://github.com/ruping/Breakpointer

LR_Gapcloser: a tiling path-based gap closer that uses long reads to complete genome assembly

Rahul Nayak — Thu, 14 May 2020 15:09:52 -0500

LR_Gapcloser is a gap closing tool using long reads from studied species. The long reads could be downloaed from public read archive database (for instance, NCBI SRA database ) or be your own data. Then they are fragmented and aligned to scaffolds using BWA mem algorithm in BWA package. In the package, we provided a compiled bwa, so the user needn't to install bwa. LR_Gapcloser uses the alignments to find the bridging that cross the gap, and then fills the long read original sequence into the genomic gaps.

Address of the bookmark: https://github.com/CAFS-bioinformatics/LR_Gapcloser

Long read assembly workshop !

Rahul Nayak — Thu, 04 Oct 2018 17:23:18 -0500

This is a tutorial for a workshop on long-read (PacBio) genome assembly.

It demonstrates how to use long PacBio sequencing reads to assemble a bacterial genome, and includes additional steps for circularising, trimming, finding plasmids, and correcting the assembly with short-read Illumina data.

Please comment if you know any other long read addembly tutorial.

Address of the bookmark: http://sepsis-omics.github.io/tutorials/modules/cmdline_assembly_v2/

Pacasus: Correction of palindromes in long reads from PacBio and Nanopore

BioStar — Mon, 12 Nov 2018 05:26:48 -0600

Tool for detecting and cleaning PacBio / Nanopore long reads after whole genome amplification. Check the poster from the Revolutionizing Next-Generation Sequencing (2nd edition) conference in the source folder: https://github.com/swarris/Pacasus/blob/master/vib2017.pdf.

The prepint version is found on http://www.biorxiv.org/content/early/2017/08/09/173872

It uses the pyPaSWAS framework for sequence alignment (https://github.com/swarris/pyPaSWAS)

Address of the bookmark: https://github.com/swarris/Pacasus

Evaluation of genome assembly software based on long reads

BioStar — Fri, 01 Feb 2019 11:55:54 -0600

TGS technologies have been used to produce highly accurate de novo assemblies of hundreds of microbial genomes and highly contiguous reconstructions of many dozens of plant and animal genomes, enabling new insights into evolution and sequence diversity. They have also been applied to resequencing analyses, to create detailed maps of structural variations in many species. Also, these new technologies have been used to fill in many of the gaps in the human reference genome.

In this report, we compare and evaluate several genome assembly software based on TSG technology. The experimentation has been performed on 4 reference genomes and the results evaluated with the QUAST software. The 11 software that have been evaluated are: Celera Assembler , Falcon , Miniasm, Newbler , SGA Assembler, Smartdenovo, Abruijn, Ra, DBG2OLC, Spades and Cerulean. The first 8 software use only long reads, while the 3 last software can merge long and short reads