text: user4401: I have what at first sight appears to be a high-quality MAG (~10 pieces, high completion%) that I built from a hybrid assembly (Illumina + Nanopore data) from a cyanobacterium.
  Workflow:
  Quality control (BBDuk) > Hybrid assembly (Unicycler) > co-binning multiple different samples (vamb) > Quality control (CheckM, anvi'o)
  *The Nanopore data is pretty old, and therefore I'd thought Unicycler might be a safer bet rather than long-read-first assemblers, due to the higher error rate
  The same strain also has 1-2 very fragmented genomes (100s of pieces) available in NCBI at scaffold level. The reads themselves are not available, so I can't just add them to the assembly.
  Is there a way to use the available NCBI genome(s) to try to check/polish mine? (I've checked out Improve a reference genome with sequencing data but it's already a few years old so I imagine different tools might be applicable)
  Thank you for your time!
  
  user2108: If your goal is to compare to the NCBI version I'd suggest using QUAST to compare the two assemblies, supplying the NCBI assembly as the reference genome (-r option I think). This will not guarantee anything, but you would expect the two assemblies to be fairly syntenic and to have good overlap of sequences. The tool includes visualization, but you could also use an approach like circoletto or dot plots for something a little more intuitive/simpler.
  I'd expect your genome to be quite a bit higher quality even as a metagenome-derived assembly due to nanopore contiguity, I'm not sure that using the NCBI reference to improve yours is a great idea. I'd instead suggest following @M__'s suggestion to scaffold with your nanopore reads (you will have to map them onto your full metagenome first, though approach may differ based on what the scaffolder wants). I'm not up on the current methods here but here is a recent tool. If you have 10 pieces I'd expect to be able to close at least some of that using nanopore (even old).
  If you do choose to use the reference to improve, the tool that people seem to be using now is RagTag.
  
  user3848: Summary
  What I am saying in this post is there approximates to 3 "generations" of in silico gap-closers/polishes:
  
  GapFiller - amongst other "early"  generations (2012);
  medaka/racon/Nanopolish/Pilon (2014 - 2018 inclusively)
  Nextpolish/PEPPER/Apollo/Homopolish/NeuralPolish (2020 onwards).
  
  The trendy approach is combining 2. and 3. I can't guarantee it will work for cyanobacteria gap-closing, but the shear volume of algorithms dedicated to this purpose, infers they must be delivering results.
  Background
  Whats the issue with a reference genome? The key thing about many  bacteria is their ability to "dump" genes and then regain them via plasmid associated recombination. This can occur extensively within a single species. This makes reference associated assembly essentially impossible in many bacteria - particularly against the official NCBI reference genome. Hence, the official NCBI reference gene, is not like a human genome reference - which is a gold standard. With due to respect to those who have dedicated time to presenting the official bacterial reference genomes are not very useful.
  This BioStars post here demonstrates that even Mycobacteria tuberculosis would not assemble via a reference genome and TB in context is one of the most conserved genomes in bacteria, the SNP behaviour is famously limited.
  If attempting a reference genome it is worth considering the following:
  
  Any amount of genetic divergence can result genetic island phenomena and non-contiguous genomes that will make assembly difficult, therefore a complete genome as close to the target genome as possible is a good reference.
  
  
  For example, simply Blasting sections of you genome to look for a consistent reference that is close to yours. A custom database of complete genomes for your species my be useful, but just sending bits of your target onto the NCBI website will be useful. The idea is to get lots of bits of the genome matching the closest strain.
  
  
  QUAST was going to be my suggestion too.
  Mapping via e.g. Mummer4 against the reference genome as the "scaffold".
  
  Key question:
  
  The same strain also has 1-2 very fragmented genomes (100s of pieces) available in NCBI at scaffold level. The reads themselves are not available, so I can't just add them to the assembly.
  
  I'm not sure what "not available" means.
  Key suggestion: Polishing
  The other approach are recent de novo assemblers and in silico "polishing". What I'm recommending is to first try "polishing". This doesn't exclude subsequent reference genome assemblies - not at all - but it's a way to improve the de novo assemblies and if there are still issues then consider a reference genome.
  Firstly I would read someone who is really up to date, e.g.
  
  Comparative evaluation of Nanopore polishing tools for microbial genome assembly and polishing strategies for downstream analysis
  
  They are medaka fans and are using this as part of a combination approach. medaka was part of the established stable of "polishers"
  
  GapFiller - I don't think this is useful.
  racon is specifically for long reads
  Nanopolish here
  
  The authors prefer medaka then goes on to describe
  
  Nextpolish, PEPPER, Apollo, Homopolish, and NeuralPolish
  
  Much more recent from 2020 ... thats a lot of polishing tools. Note, PEPPER and Apollo are often used for algorithms names - make sure its 2020 onwards, with PEPPER being 2021. They suggest medaka x PEPPER.
  I really think this combination polishing approach with an established (medaka) and one the very latest polishing algorithms, such as PEPPER (2021) is really worth trying. If it doesn't work for the specific purpose it will certainly improve the contig quality. However, given the shear volume of work dedicated to this area then there has to be good reason.
  By the same token that
  
  Money doesnt give happiness, but sure gives a better standard of misery.
  
  Then ...
  
  Polishing might not give gap closure, but sure gives a better standard of quality to the same old bunch of miserable contigs
  
  
  de novo Assembly
  Maybe Miniasm? I'd look at "polishing" the de novo assembly you have.
  RagTag as a SPAdes alternative is interesting @MaximilianPress
  
original_text: 4401: <p>I have what at first sight appears to be a high-quality MAG (~10 pieces, high completion%) that I built from a hybrid assembly (Illumina + Nanopore data) from a cyanobacterium.</p>
  <p>Workflow:
  Quality control (BBDuk) &gt; Hybrid assembly (Unicycler) &gt; co-binning multiple different samples (vamb) &gt; Quality control (CheckM, anvi'o)</p>
  <p>*The Nanopore data is pretty old, and therefore I'd thought Unicycler might be a safer bet rather than long-read-first assemblers, due to the higher error rate</p>
  <p>The same strain also has 1-2 very fragmented genomes (100s of pieces) available in NCBI at scaffold level. The reads themselves are not available, so I can't just add them to the assembly.</p>
  <p>Is there a way to use the available NCBI genome(s) to try to check/polish mine? (I've checked out <a href="https://bioinformatics.stackexchange.com/q/97/4401">Improve a reference genome with sequencing data</a> but it's already a few years old so I imagine different tools might be applicable)</p>
  <p>Thank you for your time!</p>
   2108: <p>If your goal is to compare to the NCBI version I'd suggest using <a href="https://github.com/ablab/quast" rel="nofollow noreferrer">QUAST</a> to compare the two assemblies, supplying the NCBI assembly as the reference genome (<code>-r</code> option I think). This will not guarantee anything, but you would expect the two assemblies to be fairly syntenic and to have good overlap of sequences. The tool includes visualization, but you could also use an approach like <a href="https://academic.oup.com/bioinformatics/article-abstract/26/20/2620/194655" rel="nofollow noreferrer">circoletto</a> or <a href="http://blog.dnanexus.com/2018-01-11-dot-an-interactive-dot-plot-viewer-for-comparative-genomics#" rel="nofollow noreferrer">dot plots</a> for something a little more intuitive/simpler.</p>
  <p>I'd expect your genome to be quite a bit higher quality even as a metagenome-derived assembly due to nanopore contiguity, I'm not sure that using the NCBI reference to improve yours is a great idea. I'd instead suggest following @M__'s suggestion to scaffold with your nanopore reads (you will have to map them onto your full metagenome first, though approach may differ based on what the scaffolder wants). I'm not up on the current methods here but <a href="https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-021-04451-7" rel="nofollow noreferrer">here</a> is a recent tool. If you have 10 pieces I'd expect to be able to close at least some of that using nanopore (even old).</p>
  <p>If you do choose to use the reference to improve, the tool that people seem to be using now is <a href="https://genomebiology.biomedcentral.com/articles/10.1186/s13059-022-02823-7" rel="nofollow noreferrer">RagTag</a>.</p>
   3848: <p><strong>Summary</strong>
  What I am saying in this post is there approximates to 3 &quot;generations&quot; of <em>in silico</em> gap-closers/polishes:</p>
  <ol>
  <li>GapFiller - amongst other &quot;early&quot;  generations (2012);</li>
  <li>medaka/racon/Nanopolish/Pilon (2014 - 2018 inclusively)</li>
  <li>Nextpolish/PEPPER/Apollo/Homopolish/NeuralPolish (2020 onwards).</li>
  </ol>
  <p>The trendy approach is combining 2. and 3. I can't guarantee it will work for cyanobacteria gap-closing, but the shear volume of algorithms dedicated to this purpose, infers they must be delivering results.</p>
  <p><strong>Background</strong>
  Whats the issue with a reference genome? The key thing about <em>many</em>  bacteria is their ability to &quot;dump&quot; genes and then regain them via plasmid associated recombination. This can occur extensively within a single species. This makes reference associated assembly essentially impossible in many bacteria - <em>particularly against the official NCBI reference genome</em>. Hence, the official <em>NCBI reference gene</em>, is not like a human genome reference - which is a gold standard. With due to respect to those who have dedicated time to presenting the <em>official bacterial reference genomes</em> are not very useful.</p>
  <p>This BioStars post <a href="https://www.biostars.org/p/462167/" rel="nofollow noreferrer">here</a> demonstrates that even <em>Mycobacteria tuberculosis</em> would not assemble via a reference genome and TB in context is one of the most conserved genomes in bacteria, the SNP behaviour is famously limited.</p>
  <p>If attempting a reference genome it is worth considering the following:</p>
  <ol>
  <li>Any amount of genetic divergence can result <em>genetic island</em> phenomena and non-contiguous genomes that will make assembly difficult, therefore a complete genome as close to the target genome as possible is a good reference.</li>
  </ol>
  <ul>
  <li>For example, simply Blasting sections of you genome to look for a consistent reference that is close to yours. A custom database of complete genomes for your species my be useful, but just sending bits of your target onto the NCBI website will be useful. The idea is to get lots of bits of the genome matching the closest strain.</li>
  </ul>
  <ol start="2">
  <li>QUAST was going to be my suggestion too.</li>
  <li>Mapping via e.g. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802927/" rel="nofollow noreferrer">Mummer4</a> against the reference genome as the &quot;scaffold&quot;.</li>
  </ol>
  <p><strong>Key question:</strong></p>
  <blockquote>
  <p>The same strain also has 1-2 very fragmented genomes (100s of pieces) available in NCBI at scaffold level. The reads themselves are not available, so I can't just add them to the assembly.</p>
  </blockquote>
  <p>I'm not sure what &quot;not available&quot; means.</p>
  <p><strong>Key suggestion: Polishing</strong>
  The other approach are recent <em>de novo</em> assemblers and <em>in silico</em> &quot;polishing&quot;. What I'm recommending is to first try &quot;polishing&quot;. This doesn't exclude subsequent reference genome assemblies - <em>not at all</em> - but it's a way to improve the <em>de novo</em> assemblies and if there are still issues then consider a reference genome.</p>
  <p>Firstly I would read someone who is <em>really</em> up to date, e.g.</p>
  <blockquote>
  <p><a href="https://www.nature.com/articles/s41598-021-00178-w" rel="nofollow noreferrer">Comparative evaluation of Nanopore polishing tools for microbial genome assembly and polishing strategies for downstream analysis</a></p>
  </blockquote>
  <p>They are <a href="https://github.com/nanoporetech/medaka" rel="nofollow noreferrer">medaka</a> fans and are using this as part of a combination approach. medaka was part of the established stable of &quot;polishers&quot;</p>
  <ul>
  <li><a href="https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-13-S14-S8" rel="nofollow noreferrer">GapFiller</a> - I don't think this is useful.</li>
  <li><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411768/" rel="nofollow noreferrer">racon</a> is specifically for long reads</li>
  <li>Nanopolish <a href="https://www.nature.com/articles/nmeth.3444" rel="nofollow noreferrer">here</a></li>
  </ul>
  <p>The authors prefer medaka then goes on to describe</p>
  <blockquote>
  <p><a href="https://academic.oup.com/bioinformatics/article/36/7/2253/5645175" rel="nofollow noreferrer">Nextpolish</a>, <a href="https://www.nature.com/articles/s41592-021-01299-w" rel="nofollow noreferrer">PEPPER</a>, Apollo, Homopolish, and NeuralPolish</p>
  </blockquote>
  <p>Much more recent from 2020 ... thats a lot of polishing tools. Note, PEPPER and Apollo are often used for algorithms names - make sure its 2020 onwards, with PEPPER being 2021. They suggest medaka x PEPPER.</p>
  <p>I really think this combination polishing approach with an established (medaka) and one the very latest polishing algorithms, such as PEPPER (2021) is really worth trying. If it doesn't work for the specific purpose it will certainly improve the contig quality. However, given the shear volume of work dedicated to this area then there has to be good reason.</p>
  <p>By the same token that</p>
  <blockquote>
  <p>Money doesnt give happiness, but sure gives a better standard of misery.</p>
  </blockquote>
  <p>Then ...</p>
  <blockquote>
  <p>Polishing might not give gap closure, but sure gives a better standard of quality to the same old bunch of miserable contigs</p>
  </blockquote>
  <hr />
  <p><strong><em>de novo</em> Assembly</strong>
  Maybe <a href="https://academic.oup.com/bioinformatics/article/32/14/2103/1742895" rel="nofollow noreferrer">Miniasm</a>? I'd look at &quot;polishing&quot; the <em>de novo</em> assembly you have.</p>
  <p>RagTag as a SPAdes alternative is interesting @MaximilianPress</p>
  