Effective July 10, 2023, NCBI’s Assembly and Genome record pages now redirect to new NCBI Datasets pages. As previously announced, these updates are part of our ongoing effort to modernize and improve your user experience. NCBI Datasets is a new resource that makes it easier to find and download genome data.   

The following pages have been updated:

• The NCBI Assembly record pages now redirect to the new NCBI Datasets Genome record pages that describe assembled genomes and provide links to related NCBI tools such as Genome Data Viewer and BLAST.  
• The NCBI Genome record pages now redirect to the NCBI Datasets Taxonomy record pages that provide a taxonomy-focused portal to genes, genomes, and additional NCBI resources.   

During this transition, you will have the option to return to the legacy Genome and Assembly record pages. We will remove the legacy pages in early 2024.  

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.

mScaffolder scaffolds a genome using an existing high quality genome as the reference. It aligns the two genomes using nucmer utility from MUMmer and then orders and orients the contigs of the candidate genome guided by their alignments to the reference genome. Please send your questions and comments to mchakrab@uci.edu.

Citation https://www.nature.com/articles/s41588-017-0010-y

Address of the bookmark: https://github.com/mahulchak/mscaffolder

Take a look at the PRICE software from the DeRisi lab. Its meant to do something very similar. http://derisilab.ucsf.edu/index.php?page=software

You could also look at SSPACE (http://www.baseclear.com/landingpages/basetools-a-wide-range-of-bioinformatics-solutions/sspacev12/), ATLAS tools (http://www.hgsc.bcm.tmc.edu/content/bcm-hgsc-software), and SCARPA (http://compbio.cs.toronto.edu/hapsembler/scarpa.html).

See the PAGIT protocol: http://www.sanger.ac.uk/resources/software/pagit/

In particular, take a look at the IMAGE tool: http://genomebiology.com/2010/11/4/R41

Also SOAPdenovo has ha function for scaffolding. Not sure about ABYSS

Here there is a useful explanation of several tools.

https://bioinformaticsonline.com/search?q=scaffolding&entity_type=object&entity_subtype=bookmarks&offset=0&search_type=entities

I could be wrong, but the above answers to your hypothetical scenario appear to miss the point that you aren't interested in assembling the full genome, just the 100 kb part you're interested in. I suggest the following algorithm:

1. Start with the initial assembly C0 of the contigs you have identified as overlapping your region of interest, and the set S of reads those contigs contain. Let C = C0.

2. Repeat:
a. Identify paired-end reads (not in C) for which one or both ends align within, or extending, contigs in C.
b. Identify unpaired reads that align extending these new paired-end reads.
c. Construct a new assembly C' from C and the new reads identified in (a) and (b).
d. Trim C' so it does not extend more than 100 kb to either end of C0. Set C = C'.
e. Let S' denote the reads that contribute to C'. If S' does not contain any reads not present in S, stop. Otherwise, Set S = S'.

3. If you don't have a complete assembly of the region of interest, generate an STS for each end of each contig, probe a library for clones including these STSes, subclone these clones into a paired-end sequencing vector, and generate paired-end reads for this library; then try steps (1) and (2) again, adding these new sequencing reads to what you had before.

4. If your average sequencing depth for the region of interest exceeds 25 or so without filling all gaps, it is likely that the remaining gaps represent sequences that are not getting cloned in your sequencing vectors. Try different sequencing vectors.

This software implements a de novo, alignment free measure of sample genetic dissimilarity which operates upon raw sequencing reads. It is able to calculate the genetic dissimilarity between samples without any reference genome, and without assembling one.

Address of the bookmark: https://github.com/kdmurray91/kwip

An algorithmically novel approach to construct a genome graph representation of long-read-based de novo sequence assemblies. We then provide a proof of principle by creating a genome graph of seven ethnically-diverse human genomes.

https://f1000research.com/articles/7-1391/v1

Address of the bookmark: https://github.com/NCBI-Hackathons/NovoGraph

Part I – Basic Find Commands for Finding Files with Names
1. Find Files Using Name in Current Directory

Find all the files whose name is gene.txt in a current working directory.

# find . -name gene.txt

./gene.txt

2. Find Files Under Home Directory

Find all the files under /home directory with name gene.txt.

# find /home -name gene.txt

/home/gene.txt

3. Find Files Using Name and Ignoring Case

Find all the files whose name is gene.txt and contains both capital and small letters in /home directory.

# find /home -iname gene.txt

./gene.txt
./Gene.txt

4. Find Directories Using Name

Find all directories whose name is Gene in / directory.

# find / -type d -name Gene

/Gene

5. Find fasta Files Using Name

Find all php files whose name is gene.fasta in a current working directory.

# find . -type f -name gene.fasta

./gene.fasta

6. Find all PHP Files in Directory

Find all fasta files in a directory.

# find . -type f -name "*.fasta"

./gene.fasta
./cancer.fasta
./allgene.fasta

Part II – Find Files Based on their Permissions
7. Find Files With 777 Permissions

Find all the files whose permissions are 777.

# find . -type f -perm 0777 -print

8. Find Files Without 777 Permissions

Find all the files without permission 777.

# find / -type f ! -perm 777

9. Find SGID Files with 644 Permissions

Find all the SGID bit files whose permissions set to 644.

# find / -perm 2644

10. Find Sticky Bit Files with 551 Permissions

Find all the Sticky Bit set files whose permission are 551.

# find / -perm 1551

11. Find SUID Files

Find all SUID set files.

# find / -perm /u=s

12. Find SGID Files

Find all SGID set files.

# find / -perm /g+s

13. Find Read Only Files

Find all Read Only files.

# find / -perm /u=r

14. Find Executable Files

Find all Executable files.

# find / -perm /a=x

15. Find Files with 777 Permissions and Chmod to 644

Find all 777 permission files and use chmod command to set permissions to 644.

# find / -type f -perm 0777 -print -exec chmod 644 {} \;

16. Find Directories with 777 Permissions and Chmod to 755

Find all 777 permission directories and use chmod command to set permissions to 755.

# find / -type d -perm 777 -print -exec chmod 755 {} \;

17. Find and remove single File

To find a single file called gene.txt and remove it.

# find . -type f -name "gene.txt" -exec rm -f {} \;

18. Find and remove Multiple File

To find and remove multiple files such as .fa or .gb, then use.

# find . -type f -name "*.fa" -exec rm -f {} \;

OR

# find . -type f -name "*.gb" -exec rm -f {} \;

19. Find all Empty Files

To file all empty files under certain path.

# find /tmp -type f -empty

20. Find all Empty Directories

To file all empty directories under certain path.

# find /tmp -type d -empty

21. File all Hidden Files

To find all hidden files, use below command.

# find /tmp -type f -name ".*"

Part III – Search Files Based On Owners and Groups
22. Find Single File Based on User

To find all or single file called gene.txt under / root directory of owner root.

# find / -user root -name gene.txt

23. Find all Files Based on User

To find all files that belongs to user Rahul under /home directory.

# find /home -user rahul

24. Find all Files Based on Group

To find all files that belongs to group Developer under /home directory.

# find /home -group developer

25. Find Particular Files of User

To find all .txt files of user Rahul under /home directory.

# find /home -user rahul -iname "*.txt"

Part IV – Find Files and Directories Based on Date and Time
26. Find Last 50 Days Modified Files

To find all the files which are modified 50 days back.

# find / -mtime 50

27. Find Last 50 Days Accessed Files

To find all the files which are accessed 50 days back.

# find / -atime 50

28. Find Last 50-100 Days Modified Files

To find all the files which are modified more than 50 days back and less than 100 days.

# find / -mtime +50 –mtime -100

29. Find Changed Files in Last 1 Hour

To find all the files which are changed in last 1 hour.

# find / -cmin -60

30. Find Modified Files in Last 1 Hour

To find all the files which are modified in last 1 hour.

# find / -mmin -60

31. Find Accessed Files in Last 1 Hour

To find all the files which are accessed in last 1 hour.

# find / -amin -60

Part V – Find Files and Directories Based on Size
32. Find 50MB Files

To find all 50MB files, use.

# find / -size 50M

33. Find Size between 50MB – 100MB

To find all the files which are greater than 50MB and less than 100MB.

# find / -size +50M -size -100M

34. Find and Delete 100MB Files

To find all 100MB files and delete them using one single command.

# find / -size +100M -exec rm -rf {} \;

35. Find Specific Files and Delete

Find all .gb files with more than 10MB and delete them using one single command.

# find / -type f -name *.gb -size +10M -exec rm {} \;

Address of the bookmark: http://www.fishbrowser.org/software/P_RNA_scaffolder/#

BBSplit is a tool that bins reads by mapping to multiple references simultaneously, using BBMap. The reads go to the bin of the reference they map to best. There are also disambiguation options, such that reads that map to multiple references can be binned with all of them, none of them, one of them, or put in a special "ambiguous" file for each of them. Paired reads will always be kept together.

For example, if you had a library of something that was contaminated with e.coli and salmonella, you could do this:

bbsplit.sh in=reads.fq ref=ecoli.fa,salmonella.fa basename=out_%.fq outu=clean.fq int=t

This will produce 3 output files:
out_ecoli.fq (ecoli reads)
out_salmonella.fq (salmonella reads)
clean.fq (unmapped reads)

In this case, "int=t" means that the input file is paired and interleaved. For single-end reads you would leave that out. For paired reads in 2 files, you would do this:
bbsplit.sh in1=reads1.fq in2=reads2.fq ref=ecoli.fa,salmonella.fa basename=out_%.fq outu1=clean1.fq outu2=clean2.fq

BBSplit is available here:
https://sourceforge.net/projects/bbmap/

The sensitivity can be raised to be equivalent to BBMap with these flags: "minratio=0.56 minhits=1 maxindel=16000"

lordFAST, a novel long-read mapper that is specifically designed to align reads generated by PacBio and potentially other SMS technologies to a reference. lordFAST not only has higher sensitivity than the available alternatives, it is also among the fastest and has a very low memory footprint.

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