29 votes
Accepted

Why do some assemblers require an odd-length kmer for the construction of de Bruijn graphs?

From the manual of Velvet: it must be an odd number, to avoid palindromes. If you put in an even number, Velvet will just decrement it and proceed. the palindromes in biology are defined as ...
user avatar
  • 5,287
13 votes

Why do some assemblers require an odd-length kmer for the construction of de Bruijn graphs?

To expand on the answer above, in case it isn't clear, we show: Why palindromic sequences must be even in length Why palindromic sequences induce self-loops in a de Bruijn graph Why self loops in a ...
user avatar
  • 233
8 votes

How to get fasta alignment file from SAM/BAM file?

I am not sure what you mean by "fasta alignment file". If you mean a multi-sequence alignment (MSA) in the fasta format, you can't get that because SAM keeps pairwise alignments only and doesn't align ...
user avatar
  • 5,645
7 votes

How to make a distinction between the "classical" de Bruijn graph and the one described in NGS papers?

Several papers have made this distinction, and a few indeed use different terms to distinguish between them. For example, Kazaux et al. (2016) acknowledge that: These constraints favour the use of ...
user avatar
6 votes
Accepted

How can I improve a long-read assembly with a repetitive genome?

You can't resolve 20kb near identical repeats/segdups with 10kb reads. All you can do is to bet your luck on a few excessively long reads spanning some units by chance. For divergent copies, it is ...
user avatar
  • 5,645
6 votes
Accepted

Cufflinks Error: sort order of reads in BAMs must be the same

Just add --dta-cufflinks to your Hisat2 command, so that the output alignment file provides the attributes needed by Cufflinks (XS flags). This should do the job. ...
user avatar
  • 2,614
6 votes
Accepted

Is there a standard definition for "assembly polishing"?

In long-read assembly, "polish" refers to the step to improve the base accuracy of contig sequences. I believe the terminology was originated from the HGAP paper: The final consensus–calling ...
user avatar
  • 5,645
5 votes

Reordering scaffolds according to a reference without a genetic map

Have you tried Mauve alignment? Its pretty easy once you become familiar and has a GUI for further ease of use. Additionally there are a few online tutorials on how to re-order contigs/ scaffolds ...
user avatar
  • 925
5 votes

Is there a standard definition for "assembly polishing"?

Disclaimer, this answer is based on feeling I got from talks and papers, but I do not have any hard reference supporting it. I believe that genome polishing is a technique that was introduced for ...
user avatar
  • 5,287
5 votes
Accepted

estimate genome size: kmer-based approach from PacBio reads

I don't think there is a method that would estimate a genome size using raw long reads. The genome size estimates based on raw reads are done by fitting a model to kmer spectra (for instance ...
user avatar
  • 5,287
5 votes
Accepted

Authoritative source on human cytogenetic regions?

Do you mean cytoband coordinates? For the human genome you can find them for example at UCSC. hgdownload.cse.ucsc.edu/goldenPath/hg38/database/cytoBand.txt.gz
user avatar
  • 3,521
5 votes
Accepted

PacBio long-reads impact in transcriptome de novo assembly?

A few comments: Never use N50 as a metric especially for transcriptomes. It has some semblance of relevance for genome assembly, but all that is void for a transcriptome with inherently dynamic ...
user avatar
5 votes

Contamination on genome assembly

This could be organism-specific. We don't have a lot of info so far, so I would check a few more things: Run something like FRC_align. Check if there's a clear signal between regions flagged as ...
user avatar
4 votes
Accepted

How to calculate overall reference coverage with MUMmer?

I believe all you need to do is to run dnadiff from MUMmer. That will run a comparison and output a number of useful metrics.
user avatar
  • 156
4 votes

How to make a distinction between the "classical" de Bruijn graph and the one described in NGS papers?

In addition to the regular De Bruijn graph as depicted on the wikipedia, some implementations in bioinformatics feature additional processing. I guess the main reason figure 1 in the paper you linked (...
user avatar
4 votes
Accepted

Improve a reference genome with sequencing data

Depending on the coverage of your data and the complexity of the genome, you could either reassemble the genome de novo or run a reference guided (or reference assisted) assembly. It sounds like you'...
user avatar
4 votes

Improve a reference genome with sequencing data

One approach to this is to use whatever data you have to iteratively update the reference genome. You can keep chain files along the way so you can convert coordinates (e.g. in gff files) from the ...
user avatar
  • 952
4 votes

How to deal with heterozygosity during polishing of genome assembly based on long reads?

A few possibilities: Falcon Try falcon and falcon-unzip. These are designed exactly for your problem and your data: https://github.com/PacificBiosciences/FALCON Not Falcon If you think you have ...
user avatar
  • 952
4 votes
Accepted

Why can using more reads lead to a lower quality assembly?

The more reads you add the more errors you add into the assembly. This is because additional duplicate reads don't add nodes/edges to the de Bruijn graph, but those with errors do. By preselecting ...
user avatar
  • 19.3k
4 votes
Accepted

Why isn't there a standard method to convert GFA to JSON?

Short answer: no, there isn't a "standard" method, and there won't be. Long answer: like TAB-delimited and XML, JSON alone is not a specific format. You have to define a schema to give meanings to ...
user avatar
  • 5,645
4 votes
Accepted

Separation of mixed plasmid DNA sequences post whole-plasmid sequencing

You would expect to have high coverage, given the plasmids are short, so de novo assembly would be likely very easy. Given that each plasmid is present in different multiples, you would expect ...
user avatar
4 votes

Viral genome assembly using broad viral ngs pipeline?

Since you are already using the Broad Tools sets you can use Picard FastqToSam to make the conversion As far a clipDb I am unfamiliar with that and a quick google search and look at the trimmomatic ...
user avatar
  • 2,566
4 votes
Accepted

Genome assembly of SRR12196449 with SPAdes

Update 2: I looked into this a little more, with the various data sources. This is related in part to the answer submitted by OP juanjo75es, in addition to discussion on chat. I don't entirely ...
user avatar
4 votes
Accepted

Difference between paired-end, mate-pair and long read

They are all very different in separate regards, but they all refer to different wet-lab and sequencing protocols/technologies. First, PE (paired end) reads are typically short (50-300) reads, most ...
user avatar
4 votes
Accepted

Extract sequence context of high-degree nodes in assembly graphs

I don't have a recommendation of specific tools, but we might be able to use the GFA file itself to get at this with just bash commands. For these examples I use the linked GFA here, which is a ...
user avatar
4 votes

How to display novel genome assemblies or uncommon genome assemblies using the UCSC Genome Browser?

I found two methods: You want to visit this page for instructions for novel assemblies: http://genomewiki.ucsc.edu/index.php/Assembly_Hubs . However, before doing that I highly recommend that you ...
user avatar
  • 326
3 votes

Difference between de novo transcriptome assembly methods

It's really a misnomer to call StringTie's non-reference based mode 'de-novo.' It's still using the reference genome sequence to guide the transcript assembly, it's just not using the reference ...
user avatar
  • 1,806
3 votes

Difference between de novo transcriptome assembly methods

Your intuition is correct. stringTie is just looking at clumps of alignments and how they might relate to each other (either due to spliced alignments or proximity). Trinity is doing the more ...
user avatar
  • 19.3k
3 votes
Accepted

Verify a predicted protein in one genome in a different genome of the same species

This should be very easy to do. Here are some options: Use a tool like Exonerate or GeneWise that can align protein sequences to genomic DNA while attempting to model splice sites etc. As you said, ...
user avatar
  • 7,957
3 votes

How to make a distinction between the "classical" de Bruijn graph and the one described in NGS papers?

Let's first assume DNA only has one strand. An assembly de Bruijn graph is a subgraph of a complete de Bruijn graph. It contains a vertex u if u is a k-mer in reads; it contains an edge u->v, if u and ...
user avatar
  • 5,645

Only top scored, non community-wiki answers of a minimum length are eligible