BOL: Related items

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

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

shovill: Assemble bacterial isolate genomes from Illumina paired-end reads

BioStar — Sat, 02 Jan 2021 07:05:36 -0600

Shovill is a pipeline which uses SPAdes at its core, but alters the steps before and after the primary assembly step to get similar results in less time. Shovill also supports other assemblers like SKESA, Velvet and Megahit, so you can take advantage of the pre- and post-processing the Shovill provides with those too.

Address of the bookmark: https://github.com/tseemann/shovill

SViper: Swipe your Structural Variants called on long (ONT/PacBio) reads with short exact (Illumina) reads.

Neel — Sun, 22 Dec 2019 03:48:28 -0600

Call sviper

~$ ./sviper -s short-reads.bam -l long-reads.bam -r ref.fa -c variants.vcf -o polished_variants

This will output a polished_variants.vcf file, that contains all the refined variants.

Sometimes it is helpful to look at the polished sequence, e.g. with the IGV browser. In that case you want SViper to output the polished and aligned sequences in a bam file via the option --output-polished-bam:

~$ ./sviper -s short-reads.bam -l long-reads.bam -r ref.fa -c variants.vcf -o polished_variants --output-polished-bam

Address of the bookmark: https://github.com/smehringer/SViper

Jabba: Hybrid Error Correction for Long Sequencing Reads

Jit — Fri, 05 Jan 2018 03:58:14 -0600

Jabba is a hybrid error correction tool to correct third generation (PacBio / ONT) sequencing data, using second generation (Illumina) data.

Input

Jabba takes as input a concatenated de Bruijn graph and a set of sequences:

the de Bruijn graph should appear in fasta format with 1 entry per node, the meta information should be in the format:
>NODE
the set of sequences should be in fasta or fastq format. These sequences will be corrected (e.g. PacBio reads). The corrections will be written to a file Jabba fasta.
The output is a file in fasta format with corrections of the long reads, and additionally a file in the input format containing uncorrected reads.

https://github.com/biointec/jabba/wiki

https://almob.biomedcentral.com/articles/10.1186/s13015-016-0075-7

Address of the bookmark: https://github.com/biointec/jabba

Hercules: a profile HMM-based hybrid error correction algorithm for long reads

Jit — Mon, 20 Aug 2018 14:14:11 -0500

Choosing whether to use second or third generation sequencing platforms can lead to trade-offs between accuracy and read length. Several studies require long and accurate reads including de novo assembly, fusion and structural variation detection. In such cases researchers often combine both technologies and the more erroneous long reads are corrected using the short reads. Current approaches rely on various graph based alignment techniques and do not take the error profile of the underlying technology into account. Memory- and time- efficient machine learning algorithms that address these shortcomings have the potential to achieve better and more accurate integration of these two technologies. Results: We designed and developed Hercules, the first machine learning-based long read error correction algorithm. The algorithm models every long read as a profile Hidden Markov Model with respect to the underlying platformtextquoterights error profile. The algorithm learns a posterior transition/emission probability distribution for each long read and uses this to correct errors in these reads. Using datasets from two DNA-seq BAC clones (CH17-157L1 and CH17-227A2), and human brain cerebellum polyA RNA-seq, we show that Hercules-corrected reads have the highest mapping rate among all competing algorithms and highest accuracy when most of the basepairs of a long read are covered with short reads. Availability:

Hercules source code is available at https://github.com/BilkentCompGen/Hercules

Address of the bookmark: https://github.com/BilkentCompGen/Hercules

Trimmomatic: A flexible read trimming tool for Illumina NGS data

Jit — Fri, 15 Apr 2016 05:58:53 -0500

Paired End:

java -jar trimmomatic-0.35.jar PE -phred33 input_forward.fq.gz input_reverse.fq.gz output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

This will perform the following:

Remove adapters (ILLUMINACLIP:TruSeq3-PE.fa:2:30:10)
Remove leading low quality or N bases (below quality 3) (LEADING:3)
Remove trailing low quality or N bases (below quality 3) (TRAILING:3)
Scan the read with a 4-base wide sliding window, cutting when the average quality per base drops below 15 (SLIDINGWINDOW:4:15)
Drop reads below the 36 bases long (MINLEN:36)

More at http://www.usadellab.org/cms/?page=trimmomatic

Address of the bookmark: http://www.usadellab.org/cms/?page=trimmomatic

HASLR: a hybrid assembler which uses both second and third generation sequencing reads

BioStar — Mon, 04 May 2020 02:04:03 -0500

HASLR, a hybrid assembler which uses both second and third generation sequencing reads to efficiently generate accurate genome assemblies. Our experiments show that HASLR is not only the fastest assembler but also the one with the lowest number of misassemblies on all the samples compared to other tested assemblers. Furthermore, the generated assemblies in terms of contiguity and accuracy are on par with the other tools on most of the samples. Availability. HASLR is an open source tool available at https://github.com/vpc-ccg/haslr.

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

AfterQC: Automatic Filtering, Trimming, Error Removing and Quality Control for fastq data

Jit — Fri, 29 Jun 2018 03:26:03 -0500

Automatic Filtering, Trimming, Error Removing and Quality Control for fastq data AfterQC can simply go through all fastq files in a folder and then output three folders: good, bad and QC folders, which contains good reads, bad reads and the QC results of each fastq file/pair. Currently it supports processing data from HiSeq 2000/2500/3000/4000, Nextseq 500/550, MiniSeq...and other Illumina 1.8 or newer formats The author has reimplemented this tool in C++ with multithreading support to make it much faster. The new tool is called fastp and can be found at: https://github.com/OpenGene/fastp . If you prefer a C++ based tool, please use fastp instead. https://github.com/OpenGene/AfterQC

Address of the bookmark: https://github.com/OpenGene/AfterQC

getopts.pl file

Jit — Fri, 15 Jun 2018 04:43:03 -0500

SSPACE_longread complain for getopts.pl file.

To resolve this, download and have in SSPACED-Longreads folder.

Cheers :)