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	<title><![CDATA[BOL: Protocol for De novo Genome Assembly using Illumina Reads]]></title>
	<link>https://bioinformaticsonline.com/blog/view/42633/protocol-for-de-novo-genome-assembly-using-illumina-reads?</link>
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	<guid isPermaLink="true">https://bioinformaticsonline.com/blog/view/42633/protocol-for-de-novo-genome-assembly-using-illumina-reads</guid>
	<pubDate>Sat, 16 Jan 2021 21:42:11 -0600</pubDate>
	<link>https://bioinformaticsonline.com/blog/view/42633/protocol-for-de-novo-genome-assembly-using-illumina-reads</link>
	<title><![CDATA[Protocol for De novo Genome Assembly using Illumina Reads]]></title>
	<description><![CDATA[<p>In this protocol, we address and describe the de novo assembly method for small to medium-sized genomes.</p><p><strong>What is de novo genome assembly?<br /></strong>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 &ldquo;single ended&rdquo; as described above or &ldquo;paired end.&rdquo;</p><p><strong>Why genome assembly?</strong><br />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.</p><p><strong>Raw NGS data</strong><br />Reads can be saved as a Fasta file as text or in a FastQ file with their attributes.&nbsp;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.</p><p><strong>In a nutshell the protocol:</strong> <br />Get the sequence file(s) read from the sequencing machine (s). <br />Look at the readings - have an idea of what you have and what the standard is like. <br />If required, raw data cleanup/quality trimming. <br />Choose an adequate parameter set for assembly. <br />Assemble the data into scaffolds/contigs. <br />Examine the assembly performance and determine the efficiency of the assembly.</p><p><strong>Read Quality Control:</strong><br />Check the qualiy with fastQC.<br />Script<br />https://bioinformaticsonline.com/snippets/view/42540/install-fastqc-using-conda</p><p>Quality trimming/cleanup of read files.<br />This function trims adapters, barcodes and other contaminants from the reads.<br />Script<br />https://bioinformaticsonline.com/snippets/view/42542/trimmomatic-command</p><p><strong>Genome Assembly:</strong><br />The object of this portion of the protocol is to explain the method of assembling the reads trimmed by quality into draft contigs.</p><blockquote><p>spades.py -1 illumina_R1.fastq.gz -2 illumina_R2.fastq.gz --careful --cov-cutoff auto -o result_of_spades_assembly_all_illumina</p></blockquote><p>A significant range of short-read assemblers are available. Everyone with strengths and disadvantages of their own. <br /><em>Some of the assemblers available include:</em><br />Velvet<br />SOAP-denovo<br />MIRA<br />ALLPATHS</p><p>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.&nbsp;Few stuff you can note about the contigs you just created:&nbsp;They're the draft Contigs. Any mis-assemblies can occur.</p><p><strong>Mis-assembly checking and assembly metric tools:</strong><br />QUAST - Quality assessment tool for genome assembly http://bioinf.spbau.ru/quast<br />Mauve assembly metrics - http://code.google.com/p/ngopt/wiki/How_To_Score_Genome_Assemblies_with_Mauve<br />InGAP-SV - https://sites.google.com/site/nextgengenomics/ingap and http://ingap.sourceforge.net/<br />inGAP is also useful for finding structural variants between genomes from read mappings.</p><p><strong>Genome finishing tools:</strong><br />Semi-automated gap fillers:<br />Gap filler - http://www.baseclear.com/landingpages/basetools-a-wide-range-of-bioinformatics-solutions/gapfiller/</p><p>IMAGE (V2) - http://sourceforge.net/apps/mediawiki/image2/index.php?title=Main_Page</p><p><strong>Genome visualisers and editors:</strong><br />Artemis - http://www.sanger.ac.uk/resources/software/artemis/<br />IGV - http://www.broadinstitute.org/igv/</p><p><strong>Automated and semi automated annotation tools:</strong><br />Prokka - https://github.com/tseemann/prokka<br />RAST - http://www.nmpdr.org/FIG/wiki/view.cgi/FIG/RapidAnnotationServer<br />JCVI Annotation Service - http://www.jcvi.org/cms/research/projects/annotation-service/</p><p><strong>Frequent command use for the analysis are at:</strong></p><p>https://bioinformaticsonline.com/blog/view/38765/list-of-tools-frequently-used-while-genome-assembly<br />https://bioinformaticsonline.com/pages/view/42275/frequent-parameters-for-bioinformatics-tools</p>]]></description>
	<dc:creator>BioStar</dc:creator>
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