Human whole-genome sequencing (hWGS) is performed using the shotgun sequencing method developed during the Human Genome Project (HGP) of the 1990s and early 2000s. In short, genomic DNA (gDNA) is sheared (most often using a mechanical process) and then size-selected for fragments ~350bp in length. This size-selected material is then used as input for library construction.
Library construction can be performed with PCR amplification or using a “PCR-free” method that requires more input material. If enough material is available, PCR-free library construction is recommended to minimize possible bias introduced by the PCR amplification step. Library construction with amplification can be done using 500ng of gDNA (or even significantly less) while PCR-free library construction requires more and we recommend submitting at least 2ug of gDNA per sample.
Sequencing of the constructed libraries is most often performed using 2x150bp paired end (PE) reads on an Illumina sequencing platform (e.g. the HiSeq X). The standard amount of coverage targeted for hWGS is 30X. Although this translates into ~90 Gbases of total mappable sequencing (based on the hg19 reference genome), significantly more than 90 Gbases of total raw output is required for each sample. A total of ~100-110 Gbases of sequencing is produced when targeting 30X mappable coverage. This extra 10-20 Gbases of sequencing is produced to account for unmappable sequencing results in the form of duplicate reads, low-level contamination, and unmappable reads from PhiX control DNA that are expected to be found in sequencing done on any Illumina platform.
We have entered the era of the $1,000 genome. Currently this means it costs our suppliers about $1,000 of reagents to sequence a human genome to 30X coverage. Final pricing varies depending on required turnaround time and the volume of samples submitted.
Data is delivered as FASTQ.gz files through Globus Connect or a direct transfer into your Amazon Web Services (AWS) S3 account, if available.