Questions tagged [assembly]

Process of creating the original sequence from the read sequences that it generated during a sequencing experiment. Can refer to genome assembly, in which case the original sequence is a genome, or transcripts assembly, in which case the original sequences are RNA transcripts.

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34
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2answers
2k views

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

Why do some assemblers like SOAPdenovo2 or Velvet require an odd-length k-mer size for the construction of de Bruijn graph, while some other assemblers like ABySS are fine with even-length k-mers?
18
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3answers
653 views

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

All the long-read sequencing platforms are based on single-molecule sequencing which causes higher per-base error rates. For this reason a polishing step was added to genome assembly pipelines - ...
17
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1answer
525 views

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

I'm currently trying to assembly a genome from a rodent parasite, Nippostrongylus brasiliensis. This genome does have an existing reference genome, but it is highly fragmented. Here are some ...
14
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3answers
392 views

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

In Computer Science a De Bruijn graph has (1) m^n vertices representing all possible sequences of length n over ...
11
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5answers
301 views

Improve a reference genome with sequencing data

I have a DNA sample which I know doesn't quite match my reference genome - my culture comes from a subpopulation which has undergone significant mutation since the reference was created. From visual ...
9
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3answers
433 views

Pooling data in metagenome assembly

I have 12 human gut microbiome WGS Nextseq reads (151 bp paired end). What will be an effective strategy to assemble a metagenome? Let us say I have already filtered the fastq for quality, adapter ...
8
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2answers
920 views

Is there a standard definition for “assembly polishing”?

Is there a standard definition for "assembly polishing" in the field? Is there a standard definition for what polishing algorithms do? My understanding of "polishing" is strongly influenced by ...
8
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2answers
1k views

estimate genome size: kmer-based approach from PacBio reads

Can anyone suggest a software/method for kmer analysis using PacBio reads (RSII)? Something similar to Jellyfish, that I saw in a nice tutorial - but must be suitable for long, noisy reads. ...
7
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1answer
194 views

How to calculate overall reference coverage with MUMmer?

Is the MUMmer suite capable of calculating reference sequence coverage statistics for all query sequences collectively? It would be possible to achieve by parsing the output of ...
7
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1answer
316 views

wtdbg2: practical implications of k-mer fsize and psize choice

I am using wtdbg2 2.3 to assemble a human genome (sequenced on PromethION from a cell line). I filtered out reads with low average quality, and now I am trying to determine the parameters that will ...
6
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1answer
148 views

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

I have two genome assemblies of the same non-model species, call them Assembly 1 (generated from Illumina data) and Assembly 2 (generated from PacBio data). For Assembly 1, I also have predicted ...
6
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2answers
170 views

Genome assembly from error-prone reads

I understand how to assemble genome from error-free reads. I implemented like this: Construct directed overlap graph with reads as vertices and edges as maximum overlap between two vertices. ...
5
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3answers
2k views

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

I am not talking about consensus sequence, I know how to get consensus sequence using mpileup in samtools/bcftools. As I understand , SAM/BAM files are basically sequence alignment format so it's ...
5
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2answers
760 views

Difference between de novo transcriptome assembly methods

I have been looking around (including read the original papers) to understand what is essentially the difference between StringTie in non-reference based mode (de novo) and Trinity de novo assembly. I ...
5
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3answers
65 views

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

I am experimenting with adding additional reads to the input files I'm giving SOAPdenovo2, and there comes a point where a good contig I've been watching actually stops showing up. Does anyone have a ...
5
votes
2answers
256 views

Reduce number of transcripts in a highly variable de novo transcriptome assembly

I have a de novo assembly using both multiple SRA and locally sequenced transcriptomes. I started with 270M PE reads from 9 tissues. Here are the assembly stats generated with ...
5
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1answer
86 views

Is there a way to assemble contigs starting from a specific sequence?

My work involves searching for marker genes/fragments in metagenomic databases (like the Sequence Read Archive). Once I find these sequences, I would like to know more about the neighboring genomic ...
5
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1answer
131 views

Viral Metagenomics

I am analyzing viral metagenomics data (Illumina Miseq) for the first time. I have used Ray for de novo viral genome assembly before but I haven't done metagenomics analysis before. I know that there ...
5
votes
1answer
294 views

PASA pipeline: compare experimental transcripts to the reference annotation

I would like to ask if anyone has experience in running a subset of the PASA pipeline, in particular for the reconciliation of some experimental 'transcripts' with the reference annotation. In more ...
5
votes
1answer
118 views

Find paralogs in a draft genome

We generated a (diploid, chordata, highly heterozgous) genome using PacBio and we wanted to see whether it contains lineage-specific duplications (paralogs, basically). The genome is not in Ensembl ...
5
votes
1answer
245 views

What causes the difference in total length of assembled contigs and scaffolds in SOAPdenovo2?

I use SOAPdenovo2 to assemble a large genome (4.8G) using ~20X paired-end reads. The total length of contig sizes is 6.3G while total length of scaffolds is 2.7G. Note that this is a haploid genome, ...
4
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2answers
316 views

Reordering scaffolds according to a reference without a genetic map

I am trying to reorder scaffolds of a rice species, but no genetic map is available right now. Oryza sativa Japonica is a close relative of this rice species. Mummer was used to do a whole genome ...
4
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1answer
65 views

Authoritative source on human cytogenetic regions?

I am looking for a database that would keep track of human cytogenetic regions and genomic coordinates per genome assembly. I had expected the Genome Reference Consortium to have it, or Ensembl, or ...
4
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1answer
75 views

Viral genome assembly using broad viral ngs pipeline?

I am trying to assemble RNA virus genome using Broad Viral NGS pipeline BROAD VIRAL NGS PIPELINE. I am two questions : 1) As this pipeline requires unaligned bam format as input, how do I convert ...
4
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1answer
83 views

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

I'm confused why the GFA format doesn't have a standard JSON parser. It appears to be related to a decision made several years ago, here. The objection appears to be "JSON isn't necessary, as it's ...
4
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1answer
67 views

Make ipyrad use cuda-enabled NVidia card on Ubuntu

I want to use ipyrad on a new Ubuntu machine that has an NVidia Quadro K2000 card with 384 cores. One can configure ipyrad to run on a linux cluster. Do I have any options to get ipyrad to access ...
4
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0answers
50 views

What is the current state-of-the-art in assembling hybrid transcriptomes?

We are considering attempting de novo assembly of a species transcriptomes (i.e. without a reference genome) using the combined NGS outputs of Iso-seq and Illumina. One example I saw (Li et al 2017),...
3
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2answers
146 views

Separation of mixed plasmid DNA sequences post whole-plasmid sequencing

Imagine a DNA sample containing a mixture of different intact plasmids. These samples are sequenced using either MiSeq or HiSeq sequencing. Would it possible to assemble these plasmids post-sequencing ...
3
votes
1answer
380 views

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

I am running Cufflinks for transcriptome assembly using the .bam file generated by Hisat2. I tried both bam and sorted bam files ...
3
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2answers
107 views

Contamination on genome assembly

I had a question for the community. I have a genome of a new species that has been sequenced via 150pb Illumina paired-end. To verify the quality of the assembly I used the ...
3
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1answer
182 views

Difference between genome assembly and genome sequence alignment to a reference to find structural variants

I'm trying to determine what the difference and benefits of genome assembly and genome sequence alignments are when trying to identify structural variants or transposons in populations. I've been ...
3
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1answer
50 views

How to find all WGS assemblies accessions of a species

Some background Similar to the OP of https://www.biostars.org/p/377840/, I would like to programmatically BLAST a sequence to a local database of all WGS assemblies. Since this isn't feasible for the ...
3
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1answer
78 views

Viral genome finishing

I have assembled poxvirus genome using Ray. The assembly is good. Out of several thousand contigs I got, I was able to get one scaffold using Contiguator tool, which is about 90% of my genome. I have ...
3
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1answer
140 views

How to assess the quality of assembled .fasta genome files?

I have assembled 3 .fasta files from contigs infastq format of 3 different Homo sapiens. I would like to see if the assembled ...
3
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1answer
121 views

Can the canu assembler output a fastq file of the final assembly just like HGAP4?

I have assembled some genome from Sequel PacBio data both with HGAP4 on the SMRT Link interface and using canu on the command line. The HGAP4 assembler outputs a fastq file of the final assembly such ...
3
votes
1answer
120 views

Scaffolding a genome with hybrid data

I am assembling a ~500MB genome, and have ~150x long reads and ~200x 150bp PE short reads, with a ~400bp insert size. I've done a lot of work with minimap+miniasm, and have what I think is a good set ...
3
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2answers
99 views

Merge / Reconciliate several de novo transcriptome assemblies with different kmers

I am building a De Novo transcriptome reference assembly for an eukaryotic organism for which I have a genome. I've created several assemblies with rnaSpades using different kmer sizes (19 to 69 with ...
3
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0answers
1k views

SPAdes error during assembly

I try to perform a hybrid assembly using Unicycler. Unicycler use SPAdes to assembly the illumina sequences, and the program crashes while in SPAdes. The spades.log file says something about an ...
3
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0answers
183 views

How to submit a canu job on LSF high-performance computing cluster farm?

I am currently running canu on an LSF Linux server using the following script called assemble.sh: ...
2
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2answers
154 views

Genome assembly of SRR12196449 with SPAdes

I am trying to assemble the run SRR12196449 with SPAdes. The description of their project is: This project expected to standardize a method for amplification and ...
2
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2answers
73 views

How to fragment genomes into non-overlapping sequences of differing sizes?

I have bacterial chromosomes/plasmids that I have downloaded off refseq in a multi-fasta that I would like to fragment into non-overlapping fragments of between 1 kb and 15 kb. I have tried googling ...
2
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1answer
360 views

Is nanopolish worth it since faster polishing software is available?

For Oxford Nanopore contigs produced by any long-read assembler has anyone performed any benchmarks to compare the polishing tools racon, ...
2
votes
1answer
106 views

What is the meaning of these misaligned reads in a sequencing run?

I am analyzing some SARS-CoV-2 sequencing runs abd often find read alignments like the one in the image. ...
2
votes
1answer
121 views

How is consensus alignment for OLC assembly usually implemented?

Background In C++ I want to implement a simple class that performs overlap layout consensus assembly but I can not figure out the most logical data structure to use for the consensus alignment step. ...
2
votes
1answer
94 views

Correct way to assemble reference transcriptome - what's --samples_file option in Trinity?

I have 101 samples from 9 tissues of a nonmodel species, coming either from SRA or sequenced by my lab. I want to generate a reference transcriptome with Trinity for further analysis. In order to do ...
2
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2answers
354 views

Java error when launching Pilon

I am trying to use Pilon to improve a reference based on some Illumina data I have got. So, I aligned the Illumina reads to the reference using bwa. Then, I want to use Pilon to improve the reference ...
2
votes
1answer
38 views

Gold standard benchmark

This page is claimed to contain a gold standard benchmark for viral genome assembly. https://github.com/cbg-ethz/5-virus-mix The claim is here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411778/ &...
2
votes
1answer
17 views

Can SBOL encode DNA Assembly linkers?

Where in an SBOL file could you specify the linkers that would be placed as prefixes / suffixes between parts for assembly? Lets say you have a final construct of your design and want to assemble the ...
2
votes
1answer
33 views

Assemble reads for a specific gene

I have a lot of unassembled sequencing data and I want to build phylogeny for some genes from these data. I can retrieved the sequene by mapping reads to a reference sequence but if the two species ...
2
votes
2answers
33 views

fixing chromosome with new contig

I want to fix a chromosome of around 50-100Mb (chr.fasta) for a region that is missing a few kilobases of data. I have the missing sequence with a window upstream ...