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In this tutorial you will learn how to process and de novo assemble next-generation sequencing data. The tutorial includes an overview of pairing, trimming and filtering steps that should normally be undertaken prior to assembly, and some general advice for de novo assembly. In this exercise we will use the Geneious de novo assembler. Geneious Prime also includes a number of other third party de novo assemblers including Spades, Tadpole, Velvet and MIRA. See for more information on the other assemblers. The example data provided with this tutorial are Illumina reads extracted from Sequence Read Archive (SRA) entry. These data comprise paired Illumina MiSeq reads with a raw read length of 149 bp and an expected average insert size of 350 bp.
The sequences are derived from the the genome of Escherichia coli str. MG1655 (Full genome available at ). To keep this tutorial a practical size a subset of 7800 paired reads is provided. These reads map to a 10,343 bp portion of the reference genome at coordinates 4,190,266-4,200,608. This region contains 12 complete coding sequences (CDS). This tutorial requires the BBDuk Trimmer and the MAFFT multiple aligner which are both available as plugins.
Go menu Tools → Plugins to install the BBDuk and MAFFT plugins. An overview of NGS preprocessing steps available in Geneious Prime. In this exercise we show you how to pair and trim NGS reads. Maserati vx1 vocal enhancer free download free. In this exercise we will assemble the trimmed paired data using the Geneious de novo assembler.
Importing/Pairing your NGS data An NGS sequence service provider will normally provide Illumina paired read data as two separate forward and reverse read lists in fastq format. Usually standard Illumina adapters will have been trimmed by the service provider. In most cases the fastq lists will be compressed by (.gz). Geneious can import compressed or uncompressed fastq files. If you import forward and reverse read files together via menu File → From Multiple files then Geneious will offer to pair the files and create a single paired read list. Similarly, if you drag and drop pairs of read lists into the Geneious window then you will be given the option to pair the reads during the import process. Geneious will determine the likely read technology, so you only need to set the expected insert size (the expected average insert size excluding adapters) and hit OK. NGS Trimming It is important to trim reads prior to assembly.
Incorrect low quality calls at sequence ends will potentially prevent proper assembly and increase the computation and time required to perform assembly. Geneious provides the as a plugin which can be installed via menu Tools → Plugins. BBDuk ( Decontamination Using Kmers) is a fast and accurate tool for trimming and filtering NGS reads. The plugin allows you to trim adapters using presets for Illumina adapters, trim ends by quality, trim adapters based on paired read overhangs, and discard short reads (and associated pair mate) that are trimmed to below a minimum length. The BBDuk trimmer can be accessed via menu Annotate & Predict → Trim using BBDuk. The BBDuk Minimum Quality: “Q” value is a (modified Mott algorithm).
The following table shows examples of how Q correlates to% Likelihood. Choosing an appropriate Q value will depend on the overall quality of your data. In general, trimming harder (by setting a higher Q value) will improve subsequent assembly speed and quality provided it does not trim a significant proportion of your read data. For illumina reads we recommend setting a minimum Q value of 20. Q value% Likelihood call will be correct 6 75 10 90 13 95 20 99 30 99.9 Other preprocessing steps See the last section of this tutorial if you want to see a a brief summary of other preprocessing tools available for NGS read data. Otherwise click on the following link to Exercise 1 to move to the next section of this tutorial. Exercise 2: De novo assembly of paired-end data De novo assembly of the full trimmed data set Select the list of paired trimmed reads list generated in Exercise 1, called SRR513053 subset (Trimmed), and in the Toolbar click the Align/Assemble button and choose De novo Assemble.This will open the De novo Assembler Settings window.