HPCBio Applications
Applications and Services
Please, contact us to discuss how we can be of assistance in achieving your project goals or to receive a quote for your project.
De novo Genome Assembly
HPCBio has extensive experience with de novo genome assembly using one or multiple technologies, and using multiple different software tools.
- PacBio Revio: Long reads with high accuracy. It is the technology of choice for assembly of eukaryotic and bacterial genomes.
- Illumina Novaseq: used for scaffolding existing assemblies by sequencing Hi-C and/or TellSeq libraries
- Oxford Nanopore GridION: reads 5kb to 30kb, longer if desired. Used for assembly of microbial and fungal genomes and for scaffolding of eukaryotic genomes.
- Tell-Seq libraries: linked-read Illumina-based libraries that can be used for genome assembly by barcoding long molecules of DNA, preparing shotgun libraries and then reassembling the long molecules using those barcodes. They enable estimation of genome size and complexity with Genomescope and scaffolding of PacBio assemblies.
- Hi-C libraries: these are long-range Hi-C libraries (chromosomal conformational capture) used for scaffolding of existing assemblies. These libraries are preferred for long-range scaffolding of genome assemblies.
Genotyping/Variant Calling
- Germ-line and somatic variant analysis
- Structural variant analysis
Spatial and Single Cell Transcriptomics
Single Cell 10x Genomics: Single cells or nuclei from any eukaryotic species can be processed with this technology, as long as the cells are < 40uM in diameter. Libraries for RNASeq, ATAC, or Multiome (RNASeq or ATAC) can be constructed, as well as probe-based Fixed RNA libraries for human and mouse samples. This technology begins with either a single cell or nuclei suspension, with > 70% viability for cells. It then partitions the single cells into micelles, along with an adapter molecule and a barcode enclosed within a gel bead. All the mRNAs from a single-cell within an individual micelle get barcoded with the same barcode. mRNAs are then converted to cDNAs, pooled, converted into an Illumina library and sequenced on a NovaSeq to get at least 50,000 reads per cell. Sequencing requires 28 cycles for read1 (barcode) and at least 90 cycles for read2 (cDNA). Typically this is performed on a 2x100nt or 2x150nt paired-end lane. Read more about what you can do with 10x Genomics here.
The software Cell Ranger is utilized to align the reads to the appropriate annotated genome/transcriptome and generate gene counts and other features.
Please see the 10x Submission Guidelines for important submission information, protocols, and tips.
10x Visium Spatial Transcriptomics: The 10x Visium Spatial platform allows researchers to generate spatially resolved gene expression data directly from fresh frozen tissue sections. Tissue sections are first H&E or IF stained and images captured, then the same tissue section is permeabilized to release mRNA onto capture spots that contain spatially barcoded oligos fixed to the slide. mRNAs are converted to cDNAs and then collected for dual-indexed Illumina library construction and sequencing. The H&E stained image and the spatially barcoded cDNAs are overlaid to allow visualization of the gene expression within the original tissue placement. Libraries can also be made from FFPE samples using the FFPE Visium kit.
Please see the 10x Visium page for important submission information, protocols, and tips.
Bulk Gene Expression Analysis
There are several options for RNA-Seq and small RNA libraries:
RNA-Seq, Eukaryotic species:
mRNA-enriched: these libraries are constructed by first selecting polyA+ mRNAs, converting the mRNAs to cDNA, performing adaptor ligation and amplifying for the minimum number of PCR cycles required. At least 50ng of total RNA having a RIN > 7 and free from contaminating DNA should be submitted.
rRNA-depleted: for characterization of polyA+ and polyA- transcripts, libraries can be constructed by depleting rRNA and converting the leftover RNA to cDNA, instead of capturing polyA+ mRNAs.
FFPE samples: RNA-Seq libraries can be constructed from degraded and very low amounts of RNA by first converting all the RNA to cDNA and then using specific probes to remove rRNA, or by using probes that capture specific transcripts.
IsoSeq = full-length polyA mRNAs are sequenced in the PacBio Revio with high accuracy. This is the best way to fully characterize an entire transcriptome, including all splice-variants.
RNAseq, Microbial and Metagenomic samples:
rRNA-depletion: removal of rRNA can be done with probes that recognize microbial rRNAs or with probes that remove both host and microbial rRNAs. The leftover RNA is converted to RNA-Seq libraries that are individually barcoded with Unique Dual Indexes.
Small RNA, Eukaryotic species:
Libraries are constructed from 10ng of enriched small RNA fraction or 100ng of total RNA. RNAs from 15nt to 30nt in length are enriched by size selection on a PAGE gel.
Small RNA, bacterial:
Total RNA is treated with Antarctic Phosphatase and PNK, adaptors are added and RNAs 15nt to 250nt are enriched by size selection on a PAGE gel.
Circular RNAs, double-stranded RNAs:
We have extensive experience in the construction of these libraries.
Epigenomics
Methylation Sequencing:
Whole genome methylome analysis involves an enzymatic treatment that identifies two types of DNA modifications: 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC). Alternatively, reduced-representation bisulfite sequencing libraries (RRBS) can be constructed from areas of the genome that have high CpG content. Whole genome methylome libraries are typically sequenced to a depth of 10X to 30X, while RRBS libraries typically capture only 1% of the genome so they need a much lower sequencing depth.
ATAC-Seq, Cut&Run:
We have extensive experience in the construction of these libraries. If you want to make them in your lab, we recommend discussing your application with us prior to library construction to ensure that final libraries will be compatible with the latest sequencing technology.
The critical parameters for these libraries are:
- The input DNA must be sheared to a size between 100bp and 600bp prior to pull down. We get consistent results using Covaris sonicators. Ideal sonication times need to be validated for different cell types and range from 10 min to 35 min. After sonication of a test cell suspension, please purify an aliquot of the cells and run the DNA on a 1% or 2% agarose gel with appropriately sized DNA ladder to confirm that the DNA is between 100bp and 600bp. Longer DNAs do not sequence well are most likely not efficiently enriched.
- After immunoprecipitation, we strongly recommend to confirm enrichment by performing qPCR of positive control regions relative to negative control regions before submitting DNA for library construction. A collection of ChIP-Seq data is available at the encode webpage, where the peaks can be visualized on the genome browser and primers can be designed from your genomic regions of interest. A database of primers for many common targets can be found here.
Genome Annotation
Workflow Development and Deployment
Metagenomics and Metatranscriptomics
Targeted Amplicon Analysis (16S, ITS, custom)
Grant Support
Custom Analyses
Data Management and Deposition
