The High-Throughput Genomics (HTG) Shared Resource has supported the HiSeq 2500 system since 2012. The instrument is capable of two distinct run types, the High Output Run Mode and the Rapid Run Mode. However, the HTG Shared Resource exclusively runs the instrument using the High Output option. The High Output flow cell supports both single-end (1 x 50 cycle) and paired-end (2 x 125 cycle) sequencing with an average of 220 to 270 million clusters per lane. Single-end runs require approximately two days of run time whereas paired-end runs require six days.
Sequencing experiments on the HiSeq 2500 are ordered on a per lane basis. Prior to sequencing, library quality control assays (Invitrogen Qubit dsDNA High Sensitivity Assay, Agilent ScreenTape Assay, Kapa qPCR) are performed to qualify individual libraries and normalize the library pool. Costs for this service are included as part of library preparation when the HTG Shared Resource constructs the library. Alternatively, an additional fee is charged per sample or per pre-pooled sample when researchers construct libraries within their own lab.
Frequently Asked Questions
What types of flow cells are available for the HiSeq 2500?
Two types of flow cells are currently supported at the HTG Shared Resource for sequencing on the HiSeq 2500 platform; HiSeq Single Read Cluster Kits v4 and HiSeq Paired-End Cluster Kits v4. Both of these flow cell types contain 8 lanes.
What is the average volume of sequence reads delivered by each lane on a HiSeq 2500 High Output flow cell?
Illumina High Output flow cells average 220 to 270 million reads (or read-pairs in the case of paired-end flow cells) per lane. We can guarantee these numbers when the HTG Shared Resource constructs the library. Although we highly qualify all libraries that are sequenced on the Illumina platform, we are unable to guarantee these numbers when individual researchers construct their own sequencing libraries due to variability in library quality that is outside of our control.
What run types are supported on the NovaSeq platform at the HTG Shared Resource?
The HTG Shared Resource supports 2 x 125 bp sequence runs on paired end flow cells and 1 x 50 bp runs on single-read flow cells. Please contact the HTG Shared Resource for inquiries regarding alternative sequence run types.
What options of sequencing output are available when placing a HiSeq 2500 experiment order?
Sequencing experiments on the HiSeq 2500 can be ordered through the HTG Shared Resource in increments of full lanes. Each lane yields approximately 220 to 270 million clusters that produce sequence reads.
What is the run time on a HiSeq 2500?
A 1 x 50 bp run on a HiSeq 2500 High Output flow cell l requires approximately 2 days of run time. A 2 x 125 bp run on a HiSeq 2500 High Output flow cell requires approximately 6 days of run time.
Can the HTG Shared Resource provide assistance with analysis of sequence data?
The HTG Shared Resource does not provide sequence analysis services. Please contact the Bioinformatics Shared Resource (email@example.com) for assistance.
Can I construct my own libraries for sequence analysis on the HiSeq 2500?
Libraries constructed by individual researchers can be sequenced on the HiSeq 2500. Prior to sequencing, these libraries will experience multiple quality control assays which include a Qubit dsDNA HS Assay, Agilent ScreenTape Assay, and Kapa BioSystems qPCR. Although we highly qualify all libraries that are sequenced on the Illumina platform, we are unable to guarantee the yield of sequence reads when individual researchers construct their own sequencing libraries due to variability in library quality that is outside of our control.
What quantity of a sequencing library is required for sequence analysis on a HiSeq 2500 when a researcher constructs their own libraries?
We recommend providing a volume of 10-15 L for each sequence library (or pool of libraries) at a molarity of 5 nM. We are also often able to sequence libraries that are provided at molarities down to 1 nM.
Should I be concerned if adapter dimer products are present in my sequencing library?
Adapter dimer products are able to hybridize to a HiSeq 2500 flow cell more efficiently than library molecules containing inserts. The researcher should be aware that a disproportionate volume of adapter-only reads may be present in sequence data delivered when sequencing these libraries.
How do I know if my library contains adapter dimers?
Adapter dimers exhibit a size distribution of approximately 140 to 160 bp in libraries that include dual indexed adapters. Adapter dimers in single indexed libraries are approximately 120 to 130 bp.
Can I provide custom primers to be used for sequencing my libraries on the HiSeq 2500?
Custom oligonucleotides can be provided for priming Read 1 sequencing on a HiSeq 2500 High Output flow cell. Although custom primers often result in successful sequence runs, we are unable to guarantee the yield of sequence reads or the success of the run when researchers provide custom sequencing primers.
What is the optimal insert size for libraries that are sequenced on the HiSeq 2500?
HiSeq 2500 High Output flow cells can efficiently support insert sizes that range from 20 bp to 800 bp in size. As inserts extend beyond 1000 bp, the library becomes less effective at hybridizing and efficiently amplifying on the flow cell.
How are libraries qualified prior to sequence analysis on the HiSeq 2500?
Quality control assays are performed to validate libraries prior to sequence analysis on the HiSeq 2500. These assays include the following: Qubit dsDNA High Sensitivity Assay (library concentration), Agilent ScreenTape Assay (size distribution), and Kapa BioSystems qPCR of to calculate molarities and normalize libraries prior to pooling. Costs for these quality control assays are included as part of library preparation when the HTG Shared Resource constructs the library. Alternatively, an additional fee is charged per sample when researchers construct libraries within their lab.
What length of index reads are supported on the HiSeq 2500?
The HiSeq 2500 supports 1 x 8 bp, 2 x 8 bp and 2 x 10 bp index read configurations.
What recommendations are available for sequencing a low diversity library?
Examples of low diversity libraries include bisulfite-treated DNA libraries, 16S rRNA libraries, CRISPR libraries and single amplicon libraries. In each of these cases, one or more nucleotides is significantly under-represented during each cycle of the sequence run which can negatively impact accurate base calling within a sequence lane. To overcome the impact of low diversity, a balanced library such as the Illumina PhiX v3 library, can be added to the lane at a molarity such that it will represent approximately 15-20% of the sequence reads within the lane.