What is Genome Assembly?

Genome assembly involves piecing together short DNA sequence reads generated by sequencing platforms (e.g., Illumina, PacBio, Oxford Nanopore) into longer, contiguous sequences called contigs. This can be performed as:

• De Novo Assembly: Without a reference genome.
• Reference-Guided Assembly: Using a reference genome to guide the assembly process.

Step 1: Preparing Your Data

Before starting the assembly, ensure that your raw sequencing data is high quality.

1. Input Data

   • Short Reads: Illumina sequencing generates short, accurate reads ideal for scaffolding.
   • Long Reads: PacBio and Nanopore sequencing provide long reads for resolving repetitive regions.

2. Quality Control (QC)
   Use tools like FastQC or MultiQC to assess the quality of your reads:

   fastqc reads.fastq multiqc .

   Look for issues like low-quality bases, adapter contamination, or overrepresented sequences.

3. Read Trimming and Filtering
   Trim low-quality bases and adapters using Trimmomatic or Cutadapt:

   trimmomatic PE reads_R1.fastq reads_R2.fastq trimmed_R1.fastq trimmed_R2.fastq \ ILLUMINACLIP:adapters.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36

Step 2: Choosing an Assembly Strategy

Select an assembly strategy based on your data type:

• Short-Read Assemblers:

  • SPAdes: Popular for microbial genomes.
  • Velvet: Fast for smaller genomes.

• Long-Read Assemblers:

  • Canu: Ideal for long-read datasets.
  • Flye: Versatile for small and large genomes.

• Hybrid Assemblers:

  • MaSuRCA: Combines short and long reads.
  • Unicycler: Optimized for bacterial genomes.

Step 3: Running the Assembly

3.1. SPAdes (Short-Read Assembly)

SPAdes is an excellent choice for small genomes, such as bacteria.

The output includes assembled contigs (contigs.fasta) and scaffolds (scaffolds.fasta).

3.2. Canu (Long-Read Assembly)

Canu is designed for high-error long reads from PacBio or Nanopore.

The output will be in canu_output/genome.contigs.fasta.

3.3. Hybrid Assembly with Unicycler

Unicycler combines short and long reads for improved assemblies.

Step 4: Assessing Assembly Quality

After assembly, evaluate its quality using the following tools:

1. QUAST
   QUAST generates assembly statistics, such as N50, genome size, and GC content:

   quast contigs.fasta -o quast_output

2. BUSCO
   BUSCO checks genome completeness by identifying conserved genes:

   busco -i contigs.fasta -o busco_output -l fungi_odb10 -m genome

3. Assembly Graph Visualization
   Visualize assembly graphs with Bandage:

   Bandage load assembly_graph.gfa

Step 5: Post-Assembly Steps

1. Polishing
   Improve assembly accuracy using tools like Pilon (for short reads) or Racon (for long reads).

   racon long_reads.fasta mapped_reads.sam contigs.fasta > polished_contigs.fasta

2. Scaffolding
   Link contigs into scaffolds using tools like SSPACE or Opera-LG if required.

3. Annotation
   Annotate the assembled genome using Prokka for prokaryotes or Maker for eukaryotes.

   prokka --outdir annotation_output --prefix genome contigs.fasta

Step 6: Sharing and Archiving

1. Submit to Public Repositories
   Share your assembly in databases like NCBI GenBank, ENA, or DDBJ.

2. Metadata Preparation
   Include detailed metadata for your submission, such as organism name, sequencing platform, and coverage.

Best Practices

• Always perform quality checks at each stage to ensure data integrity.
• Use multiple tools to cross-validate results when working with complex genomes.
• Document parameters and software versions for reproducibility.

Conclusion

Genome assembly is a powerful process that transforms raw sequencing data into a coherent representation of an organism’s genome. By following this step-by-step guide, you can successfully assemble genomes and uncover valuable biological insights. Whether you’re assembling a microbial genome or tackling the complexities of a eukaryotic genome, these tools and strategies will set you on the path to success.

Key Findings:

1. Chromosomal Fusion and Its Molecular Signature:

   • The fusion site is located at 2q13–2q14.1 and is characterized by degenerate telomeric sequences appearing interstitially, indicating the historical head-to-head joining of ancestral chromosomes.
   • Despite being a signature of a past fusion event, these telomeric repeats are no longer functional and have undergone sequence degradation over time.

2. Extensive Duplications in the Surrounding Genomic Region:

   • The study identifies large-scale segmental duplications flanking the fusion site, with several of these regions duplicated and scattered across multiple chromosomes.
   • These duplications are predominantly located in subtelomeric and pericentromeric regions, suggesting their role in genomic instability and chromosomal evolution.

3. Paralogous Regions and Their Evolutionary Relationships:

   • A 168-kilobase (kb) segment near the fusion site has 98%–99% sequence identity with three regions on chromosome 9 (9pter, 9p11.2, and 9q13).
   • Another 67-kb region distal to the fusion site shows a high degree of homology to sequences in chromosome 22qter.
   • Additionally, a 100-kb segment exhibits 96% sequence identity with a region in chromosome 2q11.2.

4. Comparative Genomics and Evolutionary Implications:

   • By comparing the duplicated sequences and their arrangement in primates, the researchers traced the order of duplication events leading to their present distribution.
   • The presence of specific repetitive elements within these duplicated segments serves as evolutionary markers that help infer their historical rearrangements.
   • Some of these duplicated regions are associated with chromosomal inversion breakpoints, potentially contributing to evolutionary changes in primates.
   • Recurrent structural rearrangements in these regions have been linked to human chromosomal disorders.

Conclusions and Implications:

• The findings provide valuable insights into the structural evolution of human chromosome 2, which played a crucial role in human speciation.
• Understanding these segmental duplications and their evolutionary trajectories sheds light on genomic instability, which may contribute to human genetic diseases.
• The study highlights how large-scale chromosomal rearrangements, such as fusion and duplication, have influenced the evolutionary divergence of humans from other primates.

This research advances our understanding of human genome evolution and offers a foundation for studying the effects of structural variants in genetic disorders.

Important Date & Details

Closing Date for Registration: 10 May 2015

Details of Post

Name of Post: Junior Research Fellow- 04 Posts

Pay Scale: Rs. 12,000 or 16,00+ HRA Post Graduate degree with NET (16,000+HRA) Post Graduate Degree (12,000+HRA)

Eligibility Criteria: M.Sc. in Microbiology/Marine Microbiology/ Marine Biotechnology/Biotechnology/Bioinformatics/Zoology or equivalent degree with minimum 60% marks or equivalent grade

Age Limit- Not more than 28 years

Organisation Name: Savitribai Phule Pune University
Eligibility for the post:

Selection Procedure: The selection procedure is through personal interview. No TA/DA will be paid for appearing in the interview.

How to Apply: The candidates may send their application along with CV to the Head Department of Zoology, Savitribai Phule University on or before 10 May 2015.

Installation

General Usage Guide

Data Preprocessing Guide

Specific Tool Guides:

• BBDuk
• BBMap
• BBMask
• BBMerge
• BBNorm
• CalcUniqueness
• Clumpify
• Dedupe
• Reformat
• Repair
• Seal
• Split Nextera
• Statistics
• Tadpole
• Taxonomy

https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/

Address of the bookmark: https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/

http://funprogramming.org/

Position 1: Traineeship
Monthly fellowship (in rupee): 5,000/-
No. of vacancies: Two
Required Qualification: First Class M.Sc Bioinformatics/ Biotechnology/ Botany

Position 2: Studentship
Monthly fellowship (in rupee): 5,000/-
No. of vacancies: Two
Required Qualification: M.Phil/M.Tech Bioinformatics/ Biotechnology/ any branch of Life Science students for doing their thesis work in the area of Bioinformatics.

Age limit as on 1.1.2015, 28 years. Age relaxation will be provided for SC, ST, OBC candidates as per Govt. norms.

Interested candidates may appear for walk-in-interview on 15th May 2015 at 10.30 am at JNTBGRI, Palode, Thiruvananthapuram. The candidate should report to the Office at Palode before 10.00 am.

More at http://jntbgri.res.in/news/appointment-of-two-traineeships-and-two-studentships-in-bioinformatics/

• -e Makes the line of code be executed instead of a script
• -n Forces your line to be called in a loop. Allows you to take lines from the diamond operator (or stdin)
• -p Forces your line to be called in a loop. Prints $_ at the end

• This counts the number of quotation marks in each line and prints it

  perl -ne '$cnt = tr/"//;print "$cnt\n"' inputFileName.txt

• Adds string to each line, followed by tab

  perl -pe 's/(.*)/string\t$1/' inFile > outFile

• Append a new line to each line

  perl -pe 's//\n/' all.sent.classOnly > all.sent.classOnly.sep

• Replace all occurrences of pattern1 (e.g. [0-9]) with pattern2

  perl -p -i.bak -w -e 's/pattern1/pattern2/g' inputFile

• Go through file and only print words that do not have any uppercase letters.

  perl -ne 'print unless m/[A-Z]/' allWords.txt > allWordsOnlyLowercase.txt

• Go through file, split line at each space and print words one per line.

  perl -ne 'print join("\n", split(/ /,$_));print("\n")' someText.txt > wordsPerLine.txt

• or in other words, delete every character that is not a letter, white space or line end (replace with nothing)

  perl -pne 's/[^a-zA-Z\s]*//g' text_withSpecial.txt > text_lettersOnly.txt

• perl -pne 'tr/[A-Z]/[a-z]/' textWithUpperCase.txt > textwithoutuppercase.txt;

• Print only the second column of the data when using tabular as a separator

  perl -ne '@F = split("\t", $_); print "$F[1]";' columnFileWithTabs.txt > justSecondColumn.txt

• One-Liner: Sort lines by their length

  perl -e 'print sort {length $a <=> length $b} <>' textFile

• One-Liner: Print second column, unless it contains a number

  perl">perl -lane 'print $F[1] unless $F[1] =~ m/[0-9]/' wordCounts.txt

More at http://www.bioinsilico.org/

You can also add your favourite NGS educational material, or workshop tutorial by commenting on this bookmarks for user benefit. 

Understanding the basics of genome sequencing:

Tutorial by Luke Jostins.

http://www.genetic-inference.co.uk/blog/2009/04/basics-sequencing-dna-part-1/

http://www.genetic-inference.co.uk/blog/2009/08/basics-sequencing-dna-part-2/

A window into third-generation sequencing

http://hmg.oxfordjournals.org/content/19/R2/R227.full.pdf

==============================================

NGS data analysis pipelines

• Detecting and annotating genetic variations using the HugeSeq pipeline  DOI: 10.1038/nbt.2134
• NARWHAL, a primary analysis pipeline for NGS data http://bioinformatics.oxfordjournals.org/cgi/content/abstract/28/2/284?etoc
• RseqFlow: Workflows for RNA-Seq data analysis  DOI: 10.1093/bioinformatics/btr441
• ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence  10.1186/1471-2164-12-285
• A framework for variation discovery and genotyping using next-generation DNA sequencing data  PubMed: 21478889
• SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects  DOI: 10.1186/1471-2105-12-134 Abstract: http://www.biomedcentral.com/1471-2105/12/134/abstract
• WEP: a high-performance analysis pipeline for whole-exome data http://www.biomedcentral.com/1471-2105/14/S7/S11
• DDBJ read annotation pipeline: a cloud computing-based pipeline for high-throughput analysis of next-generation sequencing data. http://www.ncbi.nlm.nih.gov/pubmed/23657089
• GATK: a Toolkit for Genome Analysis http://www.broadinstitute.org/gatk/
• Metagenomics:http://www.nbic.nl/education/nbic-phd-school/course-schedule/ngsmetagenomics/
• RNASeq:http://www.nbic.nl/education/nbic-phd-school/course-schedule/ngsrnaseq/
• Bioinformatics and Seq courses: http://www.isb-sib.ch/training/training-activities-schedule/archive-2013.html
• Variant Detection (Model organism) Advanced tutorial https://docs.google.com/document/pub?id=1CuKkKylVDb03tnN7RSWl5EUzleetn0ctjmvaidPKLxM
• Variant Detection Introductory tutorial https://docs.google.com/document/pub?id=1ZRzrjjOCvtAu3m-IKL-rbJ1f4On60dDL_IEwG7oejdI
• Microbial de novo Assembly for Illumina Data Introductory tutorial https://docs.google.com/document/pub?id=1N3AB9ptISUu4zULqe1kXpVF0BDyGb5f5yzxWSJd_WNM
• RNAseq Differential Gene Expression Introductory tutorial https://docs.google.com/document/pub?id=1KbTiBHtvHLfPRZ39AY3uriazrINA8TJzgjjwn1zPP7Y

" Please add your favourite NGS link below in comment section for the benefit of bioinformatics community ". 

Address of the bookmark: http://chagall.med.cornell.edu/NGScourse/