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

Address of the bookmark: https://github.com/cschin/Peregrine

Address of the bookmark: https://training.galaxyproject.org/training-material/topics/variant-analysis/tutorials/non-dip/tutorial.html

What Are piRNAs?

piRNAs are the largest class of small non-coding RNAs, typically 24–32 nucleotides in length. Unlike microRNAs (miRNAs) and small interfering RNAs (siRNAs), piRNAs do not rely on Dicer enzymes for maturation. Instead, they are processed from long single-stranded precursors and associate with PIWI proteins, a subclass of the Argonaute protein family.

The primary functions of piRNAs include:

1. Silencing Transposable Elements: By targeting transposons, piRNAs prevent genomic instability, particularly in germline cells.
2. Regulating Gene Expression: piRNAs modulate gene expression at transcriptional and post-transcriptional levels.
3. Epigenetic Modulation: They guide epigenetic modifications, such as DNA methylation, to specific genomic loci.

Challenges in piRNA Research

Studying piRNAs is fraught with challenges, including:

• Short Length: Their small size complicates sequencing and alignment.
• Lack of Sequence Conservation: Unlike miRNAs, piRNAs exhibit limited sequence conservation across species.
• Complex Biogenesis: The intricate pathways of piRNA generation require sophisticated computational tools to unravel.

Bioinformatics: Illuminating the World of piRNAs

Bioinformatics has emerged as an indispensable tool for studying piRNAs, facilitating their discovery, annotation, and functional analysis. Here's how bioinformatics is transforming piRNA research:

1. Identification and Annotation

The discovery of piRNAs relies on next-generation sequencing (NGS) data. Bioinformatics tools such as piRNApredictor and Piano identify piRNA clusters and predict potential targets. Databases like piRBase and piRNAdb curate information about known piRNAs, their sequences, and associated proteins.

2. Mapping and Alignment

piRNAs often originate from repetitive regions, making their alignment challenging. Tools like Bowtie and STAR handle the unique mapping requirements of piRNAs, enabling accurate identification of piRNA clusters in genomes.

3. Functional Analysis

Bioinformatics approaches predict piRNA functions by analyzing their interactions with transposons, genes, and epigenetic marks. Algorithms such as TargetFinder and RIblast explore piRNA-mRNA interactions, shedding light on regulatory networks.

4. Evolutionary Studies

piRNAs are evolutionarily diverse, reflecting their roles in species-specific genomic defense. Comparative genomics tools help trace the evolution of piRNA clusters and their associated PIWI proteins across species.

5. Epigenomic Insights

piRNAs are key players in epigenetic regulation. Bioinformatics pipelines integrate piRNA data with chromatin immunoprecipitation sequencing (ChIP-seq) and DNA methylation data to uncover their role in shaping the epigenome.

Case Study: piRNAs in Germline Integrity

One of the hallmark functions of piRNAs is the suppression of transposable elements in the germline. For example, in Drosophila melanogaster, piRNAs target retrotransposons like gypsy and copia. Bioinformatics analyses revealed that these piRNAs guide PIWI proteins to transposon-derived RNA, ensuring genome stability during gametogenesis.

Clinical Relevance of piRNAs

Recent studies suggest that piRNAs may serve as biomarkers for diseases such as cancer, infertility, and neurodegenerative disorders. For instance:

• Cancer: Dysregulated piRNA expression has been linked to tumorigenesis, making them potential targets for cancer therapies.
• Infertility: Aberrant piRNA pathways are implicated in male infertility due to their role in spermatogenesis.
• Neurodegeneration: piRNAs may regulate neuronal gene expression, highlighting their potential in neurological research.

Future Directions

The integration of bioinformatics with emerging technologies offers exciting opportunities for piRNA research:

• Single-Cell Sequencing: Unveiling cell-specific piRNA expression and function.
• Machine Learning: Predicting piRNA functions and targets with greater accuracy.
• CRISPR-Based Tools: Editing piRNA clusters to explore their roles in vivo.

Conclusion

piRNAs are the unsung guardians of the genome, safeguarding genetic material from transposable elements and contributing to gene regulation and epigenetic programming. Bioinformatics has opened the floodgates of discovery, unraveling the complexities of piRNAs and their myriad roles in biology and disease.

As we continue to decode the piRNA landscape, these small RNAs promise to unveil big secrets about genome stability, evolution, and human health, cementing their place as a fascinating frontier in molecular biology.

Again, running spades.py will show you the options:

spades.py

This produces:

SPAdes genome assembler v3.10.1

Usage: /usr/local/SPAdes-3.10.1-Linux/bin/spades.py [options] -o <output_dir>

Basic options:
-o      <output_dir>    directory to store all the resulting files (required)
--sc                    this flag is required for MDA (single-cell) data
--meta                  this flag is required for metagenomic sample data
--rna                   this flag is required for RNA-Seq data
--plasmid               runs plasmidSPAdes pipeline for plasmid detection
--iontorrent            this flag is required for IonTorrent data
--test                  runs SPAdes on toy dataset
-h/--help               prints this usage message
-v/--version            prints version

Input data:
--12    <filename>      file with interlaced forward and reverse paired-end reads
-1      <filename>      file with forward paired-end reads
-2      <filename>      file with reverse paired-end reads
-s      <filename>      file with unpaired reads
--pe<#>-12      <filename>      file with interlaced reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-1       <filename>      file with forward reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-2       <filename>      file with reverse reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-s       <filename>      file with unpaired reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-<or>    orientation of reads for paired-end library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--s<#>          <filename>      file with unpaired reads for single reads library number <#> (<#> = 1,2,..,9)
--mp<#>-12      <filename>      file with interlaced reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-1       <filename>      file with forward reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-2       <filename>      file with reverse reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-s       <filename>      file with unpaired reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-<or>    orientation of reads for mate-pair library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--hqmp<#>-12    <filename>      file with interlaced reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-1     <filename>      file with forward reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-2     <filename>      file with reverse reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-s     <filename>      file with unpaired reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-<or>  orientation of reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--nxmate<#>-1   <filename>      file with forward reads for Lucigen NxMate library number <#> (<#> = 1,2,..,9)
--nxmate<#>-2   <filename>      file with reverse reads for Lucigen NxMate library number <#> (<#> = 1,2,..,9)
--sanger        <filename>      file with Sanger reads
--pacbio        <filename>      file with PacBio reads
--nanopore      <filename>      file with Nanopore reads
--tslr  <filename>      file with TSLR-contigs
--trusted-contigs       <filename>      file with trusted contigs
--untrusted-contigs     <filename>      file with untrusted contigs

Pipeline options:
--only-error-correction runs only read error correction (without assembling)
--only-assembler        runs only assembling (without read error correction)
--careful               tries to reduce number of mismatches and short indels
--continue              continue run from the last available check-point
--restart-from  <cp>    restart run with updated options and from the specified check-point ('ec', 'as', 'k<int>', 'mc')
--disable-gzip-output   forces error correction not to compress the corrected reads
--disable-rr            disables repeat resolution stage of assembling

Advanced options:
--dataset       <filename>      file with dataset description in YAML format
-t/--threads    <int>           number of threads
                                [default: 16]
-m/--memory     <int>           RAM limit for SPAdes in Gb (terminates if exceeded)
                                [default: 250]
--tmp-dir       <dirname>       directory for temporary files
                                [default: <output_dir>/tmp]
-k              <int,int,...>   comma-separated list of k-mer sizes (must be odd and
                                less than 128) [default: 'auto']
--cov-cutoff    <float>         coverage cutoff value (a positive float number, or 'auto', or 'off') [default: 'off']
--phred-offset  <33 or 64>      PHRED quality offset in the input reads (33 or 64)
                                [default: auto-detect]

As you can see this is also a “pipeline” of tools that can be switched on or off. SPAdes takes quite a long time, so for the purposes of this practical, something like this may suffice:

spades.py -t 4 \
          -m 32 \
          -k 31,51,71 \
          --only-assembler \
          -1 miseq.1.fastq -2 miseq.2.fastq \
          --nanopore minion.fastq \
          -o hybrid_assembly

In turn, these parameters mean

• use 4 threads
• max memory is 32Gb
• use 3 kmer values to build the de bruijn graph(s) - 31, 51 and 71
• only run the assembler, not the correction algorithm (for speed)
• read 1 and read 2 of the MiSeq data
• the nanopore data
• put the output in folder “hybrid_assembly”

Following are the genome assembly tools based on string graph:

1.SGA (String Graph Assembler) https://github.com/jts/sga

Assembles large genomes from high coverage short read data. SGA is designed as a modular set of programs, which are used to form an assembly pipeline. SGA implements a set of assembly algorithms based on the FM-index. As the FM-index is a compressed data structure, the algorithms are very memory efficient. The SGA assembly has three distinct phases. The first phase corrects base calling errors in the reads. The second phase assembles contigs from the corrected reads. The third phase uses paired end and/or mate pair data to build scaffolds from the contigs. The output of this software is a PDF report that allows the properties of the genome and data quality to be visually explored. By providing more information to the user at the start of an assembly project, this software will help increase awareness of the factors that make a given assembly easy or difficult, assist in the selection of software and parameters and help to troubleshoot an assembly if it runs into problems.

2. SAGE: String-overlap Assembly of GEnomes https://github.com/lucian-ilie/SAGE2

SAGE, for de novo genome assembly. As opposed to most assemblers, which are de Bruijn graph based, SAGE uses the string-overlap graph. SAGE builds upon great existing work on string-overlap graph and maximum likelihood assembly, bringing an important number of new ideas, such as the efficient computation of the transitive reduction of the string overlap graph, the use of (generalized) edge multiplicity statistics for more accurate estimation of read copy counts, and the improved use of mate pairs and min-cost flow for supporting edge merging. The assemblies produced by SAGE for several short and medium-size genomes compared favourably with those of existing leading assemblers.

3. FSG: Fast String Graph

The new integrated assembler has been assessed on a standard benchmark, showing that fast string graph (FSG) is significantly faster than SGA while maintaining a moderate use of main memory, and showing practical advantages in running FSG on multiple threads. Moreover, we have studied the effect of coverage rates on the running times.

4.  BASE https://github.com/dhlbh/BASE

It enhances the classic seed-extension approach by indexing the reads efficiently to generate adaptive seeds that have high probability to appear uniquely in the genome. Such seeds form the basis for BASE to build extension trees and then to use reverse validation to remove the branches based on read coverage and paired-end information, resulting in high-quality consensus sequences of reads sharing the seeds. Such consensus sequences are then extended to contigs. BASE is a practically efficient tool for constructing contig, with significant improvement in quality for long NGS reads. It is relatively easy to extend BASE to include scaffolding.

5. Fermi https://github.com/lh3/fermi/

Fermi is a de novo assembler with a particular focus on assembling Illumina short sequence reads from a mammal-sized genome. In addition to the role of a typical assembler, fermi also aims to preserve heterozygotes which are often collapsed by other assemblers. Its ultimate goal is to find a minimal set of unitigs to represent all the information in raw reads.

If you want to learn about String Graph assembler, please read the following papers -

i) The Fragment Assembly String Graph - E. W. Myers

This paper describes the String Graph concept.

ii) Efficient construction of an assembly string graph using the FM-index - Jared T. Simpson and Richard Durbin

This earlier paper from Simpson and Durbin

iii) Efficient de novo assembly of large genomes using compressed data structures - Jared T. Simpson and Richard Durbin

y a number of metrics, including:

• Overall assembly length
• Number of scaffolds/contigs
• Length of longest scaffold/contig
• Scaffold/contig N50 and N90Assembly base composition, in particular percentage GC and percentage Ns
• CEGMA completeness
• Scaffold/contig length/count distribution

assembly-stats supports two widely used presentations of these values, tabular and cumulative length plots, and introduces an additional circular plot that summarises most commonly used assembly metrics in a single visualisation. Each of these presentations is generated using javascript from a common (JSON) data structure, allowing toggling between alternative views, and each can be applied to a single or multiple assemblies to allow direct comparison of alternate assemblies.

Tabular presentation allows direct comparison of exact values between assemblies, the limitations of this approach lie in the necessary omission of distributions and the challenge of interpreting ratios of values that may vary by several orders of magnitude.

Address of the bookmark: https://github.com/rjchallis/assembly-stats

Source code, executables and the testing dataset are freely available at https://github.com/mapleforest/HaploMerger2/releases/.

Address of the bookmark: https://github.com/mapleforest/HaploMerger2/releases/