Assembly and Annotation

Assembly and Annotation RNA Analysis Haploid Variant Calling Custom Analysis

Producing an assembly is often the end goal of many sequencing projects, and there is a complex landscape of assembly options that we are happy to help you navigate. Advances in long read technology on the Oxford Nanopore and PacBio platforms have made many traditional assembly methods outdated or obsolete. Meanwhile, Illumina’s per-base value has continued to improve, and the economic accessibility of Illumina has provided a plethora of high-throughput, high-quality data.

SeqCenter specializes in leveraging both read length and read accuracy to generate high quality collapsed de novo draft assemblies, with peer-reviewed, open-source software. Below are four types of automated draft assembly services we offer, with specific requirements and example methods detailing the software used for each pipeline. As with all bioinformatic analyses, SeqCenter recommends that customers ensure that a given workflow is appropriate for their organism and sample type, and to validate all results. All draft genomes should be reviewed and adjusted according to organism-specific best practices before submission or use.


PacBio Long Read Assemblies

The quality and read length of PacBio data is unmatched in the current genomic climate. We observe an average read length between 15-18kb in our PacBio datasets with about 98% of the dataset exceeding Q30 quality scores. These long, highly accurate reads generate the best assemblies that we observe at SeqCenter, as they can easily assemble through complex and repeat-rich genomic regions.

The on-instrument base detection can also capture raw methylation kinetics, which are retained in the generated BAM files. Upon assembly, we can map the raw kinetic data to the generated assembly to report genomic locations that are likely 4mC or 6mA methylation sites, relative to the final assembly.

We routinely utilize this pipeline for bacterial and fungal samples to create beautiful de novo genome assemblies and annotations. For additional information on PacBio collapsed assemblies for higher-order Eukaryotes, please contact us. Example of methods documents can be found here for prokaryotic samples and here for eukaryotic samples, detailing the software that will be used.

Requirements:

  • Data must be sequenced on the PacBio instrument.
  • Intent to retain kinetics data must be indicated prior to sequencing. This data is not retained by default.
  • Methylation calling of the 4mC and 6mA sites is limited to bacteria/fungi/yeast.
  • Genome annotation is limited to bacteria/fungi/yeast. (Custom analysis may be available for others.)

Hybrid Assemblies with Long and Short Reads

While the generated data from the PacBio instrument can create beautiful assemblies, the cost of entry is quite high and therefore less accessible to small-scale projects. To remedy this, SeqCenter offers “hybrid” or “combo” de novo assemblies which use the long reads generated from Oxford Nanopore Technologies (ONT) and the highly accurate short reads generated from Illumina. Historically, despite their comparatively lower quality, ONT reads significantly help to bridge gaps in complex assemblies by virtue of their length.

Recent advances in ONT data allow for the “super-accurate basecalling” on ONT instruments using the v10 chemistry from Oxford Nanopore. This allows us to generate reads with moderate accuracy that can be assembled to provide a backbone. The initial assembly is then polished with Illumina reads, which are aligned to the generated assembly to make base-pair corrections in the consensus and to bridge any gaps left from the initial ONT assembly. Similar to PacBio, kinetics data is captured during ONT sequencing and contains information that can be used to call 5mC sites. This data is retained within all generated BAM files from the ONT instrument. The combination of both Illumina and Oxford Nanopore technologies allows us at SeqCenter to provide you with a high-quality draft genome assembly at an accessible cost. Examples of methods documents can be found here for prokaryotic samples and here for eukaryotic samples, detailing the software that will be used.

Requirements:

  • Data must be sequenced on both the Illumina and ONT platforms.
  • Genome annotation is limited to bacteria/fungi/yeast. (Custom analysis may be available for other sample types.)

Oxford Nanopore Long Read Assemblies

For certain use cases, you may be more interested in the overall arrangement of a genome assembly and less concerned about the per-base accuracy of the assembly. This is most applicable to labs with ongoing research projects where an ancestral strain was sequenced and well documented, and the research group is particularly focused on a specific insertion, deletion, or other rearrangement. This application is also useful for preliminary hypothesis generation. A SeqCenter de novo long read draft assembly can always be complemented later with Illumina data to improve the per-base accuracy of the assembly.

Due to lower base-pair accuracy, the generated annotation data may not reflect the true functions of an organism. SeqCenter does not recommend this draft assembly option for amateur bioinformatic uses. Examples of methods documents can be found here for prokaryotic samples and here for eukaryotic samples, detailing the software that will be used.

Requirements:

  • Data must be sequenced on the ONT platform.
  • Genome annotation is limited to bacteria/fungi/yeast. (Custom analysis may be available for other sample types.)

Short Read Assemblies

Economical and highly abundant, Illumina data has utility in genome assemblies as well. However, since the reads are significantly shorter than either PacBio or Oxford Nanopore, the chance of generating a fragmented assembly is substantially higher due to an inability to assemble through complex or repeat-rich regions.

Typically, a de novo short read draft assembly will generate more than 100 contigs for an organism with a single expected chromosome. This is often sufficient for preliminary analyses and the identification of SNPs and some deletions, but cannot provide information on large indels, mobile elements, and larger structural rearrangements due to the high prevalence of gaps.

Due to these limitations, SeqCenter does not recommend this draft assembly option for amateur bioinformatic uses. Examples of methods documents can be found here for prokaryotic samples and here for eukaryotic samples, detailing the software that will be used.

Requirements:

  • Data must be sequenced through the Illumina WGS pipeline.
  • Genome annotation is limited to bacteria/fungi/yeast. (Custom analysis may be available for other sample types.)

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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Haploid Variant Calling

Mutations can be captured through whole genome sequencing and subsequent variant calling to identify SNPs, indels, and other mutations within monoclonal, haploid samples. Analysis is valid for our Illumina DNA sequencing packages and Oxford Nanopore WGS 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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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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