Haploid Variant Calling

Assembly and Annotation RNA Analysis Haploid Variant Calling Custom Analysis

SeqCenter offers variant calling for haploid genomes using the Breseq software package. This tool works by predicting mutations relative to an assembled reference file that must be provided by the customer in advance. Both fasta genome sequence file formats (nucleotide-only) and GenBank / RefSeq annotated file formats can be used as reference files; however, annotated files are preferred and return the most insightful data. Reference files are often retrieved from online repositories, such as the NCBI website. Samples must share at least 99.5% homology with the provided reference. (For bacterial genomes, 0.5% divergence can correspond to over 10,000 predicted mutations.)

The variant call output is provided in an annotated HTML file that contains mutations categorized as single nucleotide mutations (SNPs), point insertions and deletions (indels), and new junctions (for internal rearrangements). This algorithm will not be able to detect larger insertions of outside material not listed in the reference and can struggle with larger structural rearrangements, including duplications. Machine-readable VCF format is also provided.

We do not offer variant calling for diploid or further ploidy genomes as standard services at this time, but are happy to discuss options like copy number variance (CNV) analyses as a custom offering. Please contact us for more information.

Requirements:

  • Data must be sequenced through a DNA pipeline.
  • An assembly that is less than 0.5% divergent from the sample is required. Accepted formats include:
    • Files in fasta or GenBank format (.gb, .gbk, .gbff). Other formats are not accepted.
    • Assemblies uploaded to NCBI, referenced by accession number. Please verify that the correct version is specified (GenBank vs RefSeq) and referenced by the correct accession number (GCA vs GCF.)
    • Assemblies of other samples from the same order. The assembly quality will affect the variant calling, so Nanopore Combo samples are recommended.
  • Breseq assumes that samples are homogenous and haploid.
    • If a sample is expected to be polymorphic, such as in a population of a single strain, please indicate this during the order so that the parameters can be adjusted.
    • Please contact us about variant calling for non-haploid samples.

Notes on the Reference

Choosing a reference can be daunting. Ideally, the reference assembly will be very relatively close, so that the list of predicted mutations is manageable. To give an idea of scale, a bacterial sample that is 99.5% homologous to its reference can produce over 10,000 predicted mutations.

When studying a derived organism to its ancestor, it may seem obvious to choose the ancestor as the reference. This is straightforward if an assembly exists for the ancestor. If it does not, there are a few options.

  1. If the ancestor is also closely related to a published reference genome: mutations relative between the derived and parent organism can be inferred with WGS and variant calling of both organisms against the reference. Any exact mutations predicted for the ancestor can then be effectively removed from the list for the derived organism.
  2. If the ancestor is not closely related to published genomes and a simple microbe: SeqCenter offers services intended to produce closed, reference-quality de novo assemblies for haploid samples, namely Nanopore Combos and PacBio WGS with Assembly.
  3. If the ancestor is not closely related and is not a simple microbe: WGS options are limited. SeqCenter does offer collapsed assembly services for more complex organisms, but these types of genomes tend to require iterative sequencing and more fine-tuned assembly methods to produce closed reference genomes.

We do not provide assembly-to-assembly comparisons or multi sequence alignments, but many commonly used GUI-based tools can perform these, such as Galaxy, Geneious, and packages on KBase.

Additional Resources:

Assembly and Annotation

As the volume of generated sequence data continues to expand, the scientific community has a responsibility to ensure that the data accurately reflects the genomic information of the host organism. This analysis is valid for our Illumina, Oxford Nanopore, and PacBio sequencing packages for homogeneous samples, with some options for metagenomic samples.

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RNA Analysis

RNA expression data has the power to show us the active functions within a cell moments before the suspension and isolation of RNA. This analysis is valid for our Illumina RNA sequencing packages.

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Custom Analysis

The needs and demands of the scientific community rapidly adapt to innovations in technology and literature. This analysis is valid for our Illumina, Oxford Nanopore, and PacBio sequencing packages.

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Contact:
91 43rd Street, Ste. 250
Pittsburgh, PA 15201
(878) 227-4915

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Copyright 2024

Full Flowcell:

We offer full flowcell sequencing services for libraries that have been prepared outside of our lab. For each library, we will need to know the loading concentration, phiX spike-in, and run structure. Data is delivered as a complete run folder and can be demultiplexed upon request if a sample sheet is provided.

SARS-CoV-2 Sequencing:

Our SARS-CoV-2 Sequencing services include an assembly on the SARS-CoV-2 reference genome as a scaffold. These packages provide roughly 200Mbp of paired end reads (2×151 bp) as fastq files and the assembly in fasta format.

We require a minimum sample volume of 15 µL of extracted material (or 250µL for VTM samples) and recommend that only samples with Ct values below 35 are sent (because submissions are not measured for viral load before processing). We offer services for both purified RNA and samples in VTM media that require viral RNA extraction. Please see our SARS-CoV-2 Sequencing page for more detailed information.

Microbiome Sequencing:

Please see our Microbiome Sequencing page for package details, including distributables and sample requirements.

Nanopore Sequencing:

Our Oxford Nanopore sequencing packages provide reads equal to the length of input DNA, delivered as a fastq file.

For each sample, we require a minimum volume of 60 µL, and at least 40 ng/µL dsDNA quantified by Qubit or other dye-based method (100 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Long Read Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

PacBio:

Our PacBio services deliver HiFi reads with an average read length of 8-12kb for standard offerings and up to 25kb for full flowcell runs. All package names are listed by the minimum number of >Q20 reads that will be delivered. Both assembly and annotation are included with all packages.

For each sample, a minimum volume of 60 µL and at least 35 ng/µL dsDNA (quantified by Qubit or other dye-based methods) are required. SeqCenter strongly recommends utilizing our HMW DNA Extraction service for best results; however, we will also accept samples that are shipped on dry ice to avoid DNA integrity issues caused by the shipping process.

Please see our PacBio Sequencing page for more detailed information about our services.

RNA Sequencing:

We offer both rRNA depletion RNA squencing and polyA-tail enrichment mRNA sequencing. Both provide stranded, paired end reads (2x 151bp) as fastq files.

For each sample, we require a minimum volume of 20µL and at least 50ng/µL RNA. We also recommend, but do not require, that samples be DNAse treated and have a RIN score greater than 6. We also offer Basic and Intermediate RNAseq analyses, which require an annotated reference sequence.

Please see our RNA Sequencing page for more detailed information about both our RNA sequencing and analysis packages.

Illumina Sequencing:

The Illumina sequencing packages produce paired end reads (2×151 bp) delivered as fastq files.

For each sample, we require a minimum volume of 30 µL and at least 10 ng/µL dsDNA quantified by Qubit or other dye-based method (20 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Whole Genome Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

For additional information about variant calling and short read assemblies, please see our Bioinformatics page.

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