BOL: Related items

Short-read assembly using Spades !

Abhimanyu Singh — Mon, 31 Jan 2022 07:18:16 -0600

If we only had Illumina reads, we could also assemble these using the tool Spades.

You can try this here, or try it later on your own data.

Get data

We will use the same Illumina data as we used above:

illumina_R1.fastq.gz: the Illumina forward reads
illumina_R2.fastq.gz: the Illumina reverse reads

Assemble

Run Spades:

spades.py -1 illumina_R1.fastq.gz -2 illumina_R2.fastq.gz --careful --cov-cutoff auto -o spades_assembly_all_illumina

-1 is input file of forward reads
-2 is input file of reverse reads
--careful minimizes mismatches and short indels
--cov-cutoff auto computes the coverage threshold (rather than the default setting, “off”)
-o is the output directory

Results

Move into the output directory and look at the contigs:

infoseq contigs.fasta

Stacks

Jitendra Narayan — Wed, 24 Feb 2016 15:52:30 -0600

Stacks is a software pipeline for building loci from short-read sequences, such as those generated on the Illumina platform. Stacks was developed to work with restriction enzyme-based data, such as RAD-seq, for the purpose of building genetic maps and conducting population genomics and phylogeography.

More at http://catchenlab.life.illinois.edu/stacks/

Address of the bookmark: http://catchenlab.life.illinois.edu/stacks/

ALE: a Generic Assembly Likelihood Evaluation Framework for Assessing the Accuracy of Genome and Metagenome Assemblies

Neel — Tue, 26 Apr 2016 03:38:43 -0500

Assembly Likelihood Evaluation (ALE) framework that overcomes these limitations, systematically evaluating the accuracy of an assembly in a reference-independent manner using rigorous statistical methods. This framework is comprehensive, and integrates read quality, mate pair orientation and insert length (for paired-end reads), sequencing coverage, read alignment and k-mer frequency. ALE pinpoints synthetic errors in both single and metagenomic assemblies, including single-base errors, insertions/deletions, genome rearrangements and chimeric assemblies presented in metagenomes. At the genome level with real-world data, ALE identifies three large misassemblies from the Spirochaeta smaragdinae finished genome, which were all independently validated by Pacific Biosciences sequencing. At the single-base level with Illumina data, ALE recovers 215 of 222 (97%) single nucleotide variants in a training set from a GC-rich Rhodobacter sphaeroides genome. Using real Pacific Biosciences data, ALE identifies 12 of 12 synthetic errors in a Lambda Phage genome, surpassing even Pacific Biosciences' own variant caller, EviCons. In summary, the ALE framework provides a comprehensive, reference-independent and statistically rigorous measure of single genome and metagenome assembly accuracy, which can be used to identify misassemblies or to optimize the assembly process.

More at http://www.ncbi.nlm.nih.gov/pubmed/23303509

Address of the bookmark: http://sc932.github.io/ALE/about.html

Stampy

Abhi — Fri, 20 May 2016 19:13:32 -0500

Stampy is a package for the mapping of short reads from illumina sequencing machines onto a reference genome. It's recommended for most workflows, including those for genomic resequencing, RNA-Seq and Chip-seq. Stampy excels in the mapping of reads containing that contain sequence variation relative to the reference, in particular for those containing insertions or deletions.

Address of the bookmark: http://www.well.ox.ac.uk/project-stampy

PHYMMBL

Jit — Mon, 10 Oct 2016 08:56:34 -0500

Metagenomics sequencing projects collect samples of DNA from uncharacterized environments that may contain hundreds or even thousands of species. One of the main challenges in analyzing a metagenome is phylogenetic classification of raw sequence reads into groups representing the same or similar species. Such classification is a useful prerequisite for genome assembly and for analysis of the biological diversity present in a sample. The newest sequencing technologies have simultaneously made metagenomics easier, by making the sequencing process faster, and more difficult, by producing shorter read lengths than previous technologies. Methods for classifying sequences as short as 100 base pairs (bp) have until now been relatively inaccurate, requiring metagenomics projects to use older, long-read technologies. Phymm, a new classification approach for metagenomics data which uses interpolated Markov models (IMMs) to taxonomically classify DNA sequences, can accurately classify reads as short as 100 bp. Its accuracy for short reads represents a significant leap forward over previous composition-based classification methods. PhymmBL (rhymes with "thimble"), the hybrid classifier included in this distribution which combines analysis from both Phymm and BLAST, produces even higher accuracy.

Address of the bookmark: http://www.cbcb.umd.edu/software/phymm/

YAHA

Jit — Fri, 20 Jan 2017 05:38:05 -0600

YAHA, a fast and flexible hash-based aligner. YAHA is as fast and accurate as BWA-SW at finding the single best alignment per query and is dramatically faster and more sensitive than both SSAHA2 and MegaBLAST at finding all possible alignments. Unlike other aligners that report all, or one, alignment per query, or that use simple heuristics to select alignments, YAHA uses a directed acyclic graph to find the optimal set of alignments that cover a query using a biologically relevant breakpoint penalty. YAHA can also report multiple mappings per defined segment of the query. We show that YAHA detects more breakpoints in less time than BWA-SW across all SV classes, and especially excels at complex SVs comprising multiple breakpoints.

Availability: YAHA is currently supported on 64-bit Linux systems. Binaries and sample data are freely available for download from http://faculty.virginia.edu/irahall/YAHA.

Contact:

http://genome.wustl.edu/people/groups/detail/hall-lab/

Address of the bookmark: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463118/

Multi-metagenome assembly

Radha Agarkar — Fri, 03 Mar 2017 10:14:18 -0600

This project contains scripts and tutorials on how to assemble individual microbial genomes from metagenomes, as described in:

Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes

Mads Albertsen, Philip Hugenholtz, Adam Skarshewski, Gene W. Tyson, Kåre L. Nielsen and Per .H. Nielsen

Nature Biotechnology 2013, doi: 10.1038/nbt.2579

Address of the bookmark: https://github.com/MadsAlbertsen/multi-metagenome

Meraculous: De Novo Genome Assembly with Short Paired-End Reads

Jit — Tue, 07 Nov 2017 04:36:10 -0600

We describe a new algorithm, meraculous, for whole genome assembly of deep paired-end short reads, and apply it to the assembly of a dataset of paired 75-bp Illumina reads derived from the 15.4 megabase genome of the haploid yeast Pichia stipitis. More than 95% of the genome is recovered, with no errors; half the assembled sequence is in contigs longer than 101 kilobases and in scaffolds longer than 269 kilobases. Incorporating fosmid ends recovers entire chromosomes. Meraculous relies on an efficient and conservative traversal of the subgraph of the k-mer (deBruijn) graph of oligonucleotides with unique high quality extensions in the dataset, avoiding an explicit error correction step as used in other short-read assemblers. A novel memory-efficient hashing scheme is introduced. The resulting contigs are ordered and oriented using paired reads separated by ∼280 bp or ∼3.2 kbp, and many gaps between contigs can be closed using paired-end placements. Practical issues with the dataset are described, and prospects for assembling larger genomes are discussed.

Address of the bookmark: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158087/

NxRepair: error correction in de novo assemblies using Nextera Mate Pair Reads

BioStar — Thu, 24 Jan 2019 10:35:12 -0600

NxRepair is a python module that automatically detects large structural errors in de novo assemblies using Nextera mate pair reads. The decector will break a contig at the site of an identified misassembly and will generate a new fasta file containing both the corrected contigs and the correct, unaffected contigs.

https://nxrepair.readthedocs.io/en/latest/tutorial.html

nxrepair aligned_matepairs.bam assemblyfasta.fasta error_locations.csv new_fasta.fasta

Address of the bookmark: https://github.com/rebeccaroisin/nxrepair

Understanding HiFi Reads !

Rahul Nayak — Thu, 24 Mar 2022 19:48:11 -0500

While little public data is available for either of the new synthetic long read approaches, Illumina showed an example comparison earlier this year at the Festival of Genomics & Biodata conference (FoG 2022). In the IGV screenshot presented (below), synthetic Infinity reads – labeled “Longas” – are at the top, followed by standard Illumina short reads, and PacBio HiFi reads labeled “CCS” depicted at the bottom:

Address of the bookmark: http://pacb.com/blog/the-hifi-difference-true-long-reads-vs-synthetic-long-reads/