followed by ligation of Illumina-specific adaptor oligonucleotides.
To begin constructing the cDNA library, the samples are first
fragmented and random primers are added. Following first and
second strand synthesis, the adaptors are ligated. The samples are
then processed with bead-based DNA size selection steps to
remove unligated adaptor. To increase speed and efficiency during
these steps, it is easiest to have samples arrayed in 96-well PCR
plates. This setup enables DNA size selection to be performed
using a multichannel pipette, giving less time for beads to dry out
and negatively affect library yield.
To determine an accurate quantification of the quality and
quantity of our cDNA samples (steps 3 and 4), we use the Kapa
library quantification kits for Illumina sequencing platforms, as well
as measurements from the Qubit assay and the bioanalyzer assay to
inform the multiplexed pooling of sample libraries (step 5). The
Kapa quantification kit measures the abundance of fragments
tagged by the Illumina-specific sequencing adapter oligos. This
provides a more accurate assessment of the concentration of
adaptor-tagged library detectable by Illumina sequencing instruments, whereas a Nanodrop Spectrophotometer would quantify all
of the DNA. A bioanalyzer assay is also a useful measurement of
sample quality, which enables detection of unligated Illumina
adapter fragments. Sequencing centers often perform an additional
quantification test by running submitting samples through a bioanalyzer system to confirm previous Kapa Quantification results.
We will describe how to perform a sequencing run on the
NextSeq 500 (steps 6 and 7). In a sequencing run, the number
of cycles performed in a read is one more cycle than the number of
cycles analyzed. For example, a paired-end 150-cycle run performs
reads of 151-cycles (2 Â 151) for a total of 302 cycles. At the end of
the run, 2 Â 150 cycles are analyzed. The extra cycle is required for
phasing and prephasing calculations. After the run, an instrument
wash begins automatically using components already loaded on the
instrument.
Cluster generation and sequencing are performed on instrument. During cluster generation, single DNA molecules are bound
to the surface of the flow cell and then amplified to form clusters.
Clusters are imaged using two-channel sequencing chemistry and
filter combinations specific to each of the fluorescently labeled
chain terminators. After imaging of a tile on the flow cell is complete, the next tile is imaged. The process is repeated for each cycle
of sequencing. Following image analysis, the software performs
base calling, filtering, and quality scoring. Run progress and statistics can be monitored in the following locations: The NCS interface, BaseSpace Sequence Hub, Local Run Manager, or networked
computer using the Sequencing Analysis Viewer (SAV) software.
We will describe how to perform the sequencing run using the
RNAseq Workflow
485
To begin constructing the cDNA library, the samples are first
fragmented and random primers are added. Following first and
second strand synthesis, the adaptors are ligated. The samples are
then processed with bead-based DNA size selection steps to
remove unligated adaptor. To increase speed and efficiency during
these steps, it is easiest to have samples arrayed in 96-well PCR
plates. This setup enables DNA size selection to be performed
using a multichannel pipette, giving less time for beads to dry out
and negatively affect library yield.
To determine an accurate quantification of the quality and
quantity of our cDNA samples (steps 3 and 4), we use the Kapa
library quantification kits for Illumina sequencing platforms, as well
as measurements from the Qubit assay and the bioanalyzer assay to
inform the multiplexed pooling of sample libraries (step 5). The
Kapa quantification kit measures the abundance of fragments
tagged by the Illumina-specific sequencing adapter oligos. This
provides a more accurate assessment of the concentration of
adaptor-tagged library detectable by Illumina sequencing instruments, whereas a Nanodrop Spectrophotometer would quantify all
of the DNA. A bioanalyzer assay is also a useful measurement of
sample quality, which enables detection of unligated Illumina
adapter fragments. Sequencing centers often perform an additional
quantification test by running submitting samples through a bioanalyzer system to confirm previous Kapa Quantification results.
We will describe how to perform a sequencing run on the
NextSeq 500 (steps 6 and 7). In a sequencing run, the number
of cycles performed in a read is one more cycle than the number of
cycles analyzed. For example, a paired-end 150-cycle run performs
reads of 151-cycles (2 Â 151) for a total of 302 cycles. At the end of
the run, 2 Â 150 cycles are analyzed. The extra cycle is required for
phasing and prephasing calculations. After the run, an instrument
wash begins automatically using components already loaded on the
instrument.
Cluster generation and sequencing are performed on instrument. During cluster generation, single DNA molecules are bound
to the surface of the flow cell and then amplified to form clusters.
Clusters are imaged using two-channel sequencing chemistry and
filter combinations specific to each of the fluorescently labeled
chain terminators. After imaging of a tile on the flow cell is complete, the next tile is imaged. The process is repeated for each cycle
of sequencing. Following image analysis, the software performs
base calling, filtering, and quality scoring. Run progress and statistics can be monitored in the following locations: The NCS interface, BaseSpace Sequence Hub, Local Run Manager, or networked
computer using the Sequencing Analysis Viewer (SAV) software.
We will describe how to perform the sequencing run using the
RNAseq Workflow
485