BOL: Related items

Genome assembly tutorial "Genome Assembly for short and long reads"

Jit — Sat, 19 Jan 2019 17:29:53 -0600

In this lab we will perform de novo genome assembly of a bacterial genome. You will be guided through the genome assembly starting with data quality control, through to building contigs and analysis of the results. At the end of the lab you will know:

How to perform basic quality checks on the input data
How to run a short read assembler on Illumina data
How to run a long read assembler on Pacific Biosciences or Oxford Nanopore data
How to improve the accuracy of a long read assembly using short reads
How to assess the quality of an assembly

https://bioinformaticsdotca.github.io/high-throughput_biology_2017

Address of the bookmark: https://bioinformaticsdotca.github.io/high-throughput_biology_2017_module6_lab

LACHESIS: Genome Assembly with Hi-C-based Contact Probability Maps (LACHESIS)

Jit — Mon, 14 May 2018 04:26:30 -0500

LACHESIS is method that exploits contact probability map data (e.g. from Hi-C) for chromosome-scale de novo genome assembly.

Further information about LACHESIS, including source code, documentation and a user's guide are available at: http://shendurelab.github.io/LACHESIS.

Manuscript describing LACHESIS was published as: Burton JN#, Adey A, Patwardhan RP, Qiu R, Kitzman JO, Shendure J#. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nature Biotechnology 2013 Dec;31(12):1119-25. doi: 10.1038/nbt.272. PubMed PMID: 24185095.

http://shendurelab.github.io/LACHESIS/

Address of the bookmark: http://shendurelab.github.io/LACHESIS/

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

fragScaff: Genome Assembly with Contiguity Preserving Transposition

Jit — Mon, 14 May 2018 04:28:14 -0500

Contiguity preserving transposition and sequencing (CPT-seq) is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to >1 megabase. This software, fragScaff, leverages coincidences between the content of different pools as a source of contiguity information for scaffolding de novo genome assemblies. FragScaff is complementary to Lachesis, providing midrange contiguity to support robust, accurate chromosome-scale de novo genome assemblies without the need for laborious in vivo cloning steps.

Further information about fragScaff, including source code, is available at:https://sourceforge.net/projects/fragscaff/files.

Manuscript describing fragScaff was published as: Adey A, Kitzman JO, Burton JN, Daza R, Kumar A, Christiansen L, Ronaghi M, Amini S, L Gunderson K, Steemers FJ, Shendure J#. In vitro, long-range sequence information for de novo genome assembly via transposase contiguity. Genome Research 2014 Dec;24(12):2041-9. doi: 10.1101/gr.178319.114. PubMed PMID: 25327137.

Address of the bookmark: https://sourceforge.net/projects/fragscaff/files/

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

HASLR: a tool for rapid genome assembly of long sequencing reads

LEGE — Fri, 31 Jan 2020 05:50:15 -0600

HASLR is a tool for rapid genome assembly of long sequencing reads. HASLR is a hybrid tool which means it requires long reads generated by Third Generation Sequencing technologies (such as PacBio or Oxford Nanopore) together with Next Generation Sequencing reads (such as Illumina) from the same sample.

Address of the bookmark: https://github.com/vpc-ccg/haslr

Biological databases !

BioStar — Wed, 12 Feb 2020 01:16:29 -0600

Now a days there are a lots of genomics databases available around the world. This bookmark is created to provide all links in one place ...

ftp://ftp.ncbi.nih.gov/genomes/

https://hgdownload.soe.ucsc.edu/downloads.html

Address of the bookmark: ftp://ftp.ncbi.nih.gov/genomes/

PBSuite: Software for Long-Read Sequencing Data from PacBio

Jit — Mon, 27 Feb 2017 09:54:47 -0600

PBJelly - the genome upgrading tool.
PBHoney - the structural variation discovery tool

Both are contained within the PBSuite code found in downloads.

----- PBJelly -----
Read The Paper
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047768

PBJelly is a highly automated pipeline that aligns long sequencing reads (such as PacBio RS reads or long 454 reads in fasta format) to high-confidence draft assembles. PBJelly fills or reduces as many captured gaps as possible to produce upgraded draft genomes.

----- PBHoney -----
Read The Paper
http://www.biomedcentral.com/1471-2105/15/180/abstract

PBHoney is an implementation of two variant-identification approaches designed to exploit the high mappability of long reads (i.e., greater than 10,000 bp). PBHoney considers both intra-read discordance and soft-clipped tails of long reads to identify structural variants.

Address of the bookmark: https://sourceforge.net/projects/pb-jelly/

CABOG: Celera Assembler with Best Overlap Graph

Abhimanyu Singh — Mon, 15 May 2017 05:04:39 -0500

CABOG (Celera Assembler with Best Overlap Graph) is scientific software for DNA research. CABOG has been a critical component of many genome sequencing projects. CABOG operates on small genomes such as bacterial as well as large genomes such as mammalian. CABOG is an extension of the Celera Assembler software that was originally developed at Celera for the 2001 publication of the first draft human genome sequence. The software was released to the public domain in 2004. Its open source repository on Source Forge is an internet resource for scientists around the world.

CABOG is one of many software programs called genome assemblers. These programs exist to overcome the fundamental limitation of all sequencing machines, namely, that they read out very few DNA letters at a time. These programs reconstruct genomes that are billions of letters long from the hundreds of letters per read that modern sequencers provide. What these programs do is often described as a scaled up version of a family solving a jigsaw puzzle.

The CABOG software was the first to accomplish many scientific goals. It was the first to assemble the genome of a multicellular organism (Drosophila melanogaster, 2000). It was the first to assemble both parental haplotypes of one human genome (J. Craig Venter, 2007). It was the first to assemble environmental sequence from the oceans (Sargasso Sea in 2004 and Global Ocean Sampling in 2007). It was first to combine reads from first-generation Sanger sequencing machines and second-generation pyrosequencing machines (Marine microbes, 2006). Today, CABOG is one of the leading assembly programs for data sets that include paired end data from the Roche 454 line of sequencing machines.

Address of the bookmark: http://www.jcvi.org/cms/research/projects/cabog/overview/

Protocol for De novo Genome Assembly using Illumina Reads

BioStar — Sat, 16 Jan 2021 21:42:11 -0600

In this protocol, we address and describe the de novo assembly method for small to medium-sized genomes.

What is de novo genome assembly?
The method of taking a large number of short DNA sequences and placing them back together to create a reflection of the original chromosomes from which the DNA originated relates to genome assembly. No previous knowledge of the source DNA sequence length, structure or composition is inferred by De novo genome assemblies. The DNA of the target organism is split up into millions of tiny parts and read on a sequencing computer in a genome sequencing experiment. Depending on the sequencing system used, these "reads" range from 20 to 1000 nucleotide base pairs (bp) in length. Usually, length reads of 36 - 150 bp are produced for Illumina style short read sequencing. These reads can be either “single ended” as described above or “paired end.”

Why genome assembly?
In basic research into why and how they live, as well as in applied topics, identifying the DNA sequence of an organism is useful. Awareness of a DNA sequence may be useful in virtually any biological research because of the relevance of DNA to living things. For example, it may be used in medicine to classify, diagnose and eventually improve genetic disorder therapies. Similarly, pathogens study can lead to treatments for infectious diseases.

Raw NGS data
Reads can be saved as a Fasta file as text or in a FastQ file with their attributes. FastQ is the most common read file format since this is what the Illumina sequencing pipeline creates. This will henceforth be the subject of our conversation.

In a nutshell the protocol:
Get the sequence file(s) read from the sequencing machine (s).
Look at the readings - have an idea of what you have and what the standard is like.
If required, raw data cleanup/quality trimming.
Choose an adequate parameter set for assembly.
Assemble the data into scaffolds/contigs.
Examine the assembly performance and determine the efficiency of the assembly.

Read Quality Control:
Check the qualiy with fastQC.
Script
https://bioinformaticsonline.com/snippets/view/42540/install-fastqc-using-conda

Quality trimming/cleanup of read files.
This function trims adapters, barcodes and other contaminants from the reads.
Script
https://bioinformaticsonline.com/snippets/view/42542/trimmomatic-command

Genome Assembly:
The object of this portion of the protocol is to explain the method of assembling the reads trimmed by quality into draft contigs.

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

A significant range of short-read assemblers are available. Everyone with strengths and disadvantages of their own.
Some of the assemblers available include:
Velvet
SOAP-denovo
MIRA
ALLPATHS

Next step is to assess the suitability and what to do with a draft package of contiguous details for the remainder of the study now. Few stuff you can note about the contigs you just created: They're the draft Contigs. Any mis-assemblies can occur.

Mis-assembly checking and assembly metric tools:
QUAST - Quality assessment tool for genome assembly http://bioinf.spbau.ru/quast
Mauve assembly metrics - http://code.google.com/p/ngopt/wiki/How_To_Score_Genome_Assemblies_with_Mauve
InGAP-SV - https://sites.google.com/site/nextgengenomics/ingap and http://ingap.sourceforge.net/
inGAP is also useful for finding structural variants between genomes from read mappings.

Genome finishing tools:
Semi-automated gap fillers:
Gap filler - http://www.baseclear.com/landingpages/basetools-a-wide-range-of-bioinformatics-solutions/gapfiller/

IMAGE (V2) - http://sourceforge.net/apps/mediawiki/image2/index.php?title=Main_Page

Genome visualisers and editors:
Artemis - http://www.sanger.ac.uk/resources/software/artemis/
IGV - http://www.broadinstitute.org/igv/

Automated and semi automated annotation tools:
Prokka - https://github.com/tseemann/prokka
RAST - http://www.nmpdr.org/FIG/wiki/view.cgi/FIG/RapidAnnotationServer
JCVI Annotation Service - http://www.jcvi.org/cms/research/projects/annotation-service/

Frequent command use for the analysis are at:

https://bioinformaticsonline.com/blog/view/38765/list-of-tools-frequently-used-while-genome-assembly
https://bioinformaticsonline.com/pages/view/42275/frequent-parameters-for-bioinformatics-tools