Illumina NextSeq 500/550 v2.5 Sequencing Kit

Product Name:Illumina NextSeq 500/550 v2.5 Sequencing Kit(150 cycles) In stock supply

Product Code: 20024904

Product brand: Illumina/Imena USA

Illumina NextSeq 500/550 sequencing kit in stock， Illumina sequencing kit, available in stock, welcome to inquire

Illumina NextSeq 500/550 sequencing kit in stockIllumina sequencing kit, available in stock, welcome to inquire

Illumina sequencing kit, available in stock, welcome to inquire

Lumina second-generation sequencing technology, synthesis sequencing at the same time (SBS), is a widely used next-generation sequencing (NGS) technology responsible for generating over 90% of the world's sequencing data.

Illumina's second-generation sequencing technology is called Sequencing by Synthesis (SBS). SBS mainly includes three steps: base extension, fluorescence excitation and signal collection, and splicing. These three steps can be repeated in a loop. Measure one base per cycle. Cycle as many times as you want to determine the number of bases (i.e. read length). The number of sequencing cycles can be set in the software. The appropriate length of sequencing depends on the nature and requirements of the project. In most cases, SR experiments (single ended sequencing) measure 1x50 bases, while PE experiments (double ended sequencing) measure 2x150 bases.

1 base extension

The dNTPs used in Illumina sequencing reagents have two modifying groups: a fluorescent group and a reversible terminating group. A. The four bases G, C, and T respectively label four different fluorescent groups, which can emit fluorescence of different wavelengths (i.e. colors) under laser excitation at different wavelengths. The fluorescence color corresponds one-to-one with the type of base, so bases can be identified based on the wavelength of the excitation light. The termination groups of the four bases are the same. Due to the presence of termination groups, all dNTPs can only extend one base at a time, regardless of the given reaction time. Moreover, since the termination group is reversible, after the fluorescence signal is collected, a shearing agent can be used to remove the termination group, restoring the base to its natural and extensible state. At this point, the next cycle can be continued and another base can be measured.

The reversible termination group technology is a major feature of Illumina second-generation sequencing. It, together with bridge PCR technology, constitutes the two pillars of Illumina second-generation sequencing, laying a solid foundation for stable acquisition of high-quality sequencing data.

2 Signal acquisition

The HiSeq series sequencer has two laser tubes, red and green, and two color filters. They can be combined in pairs to form four different excitation wavelengths, which are precisely used to excite the A, G, C, and T bases.

The signal generated by the fluorescent groups labeled on the cluster under laser excitation can be collected by capturing or scanning with a camera. The scanning technology is relatively fast and has been adopted by the X10 series. Due to the large area of FC and the need to take separate photos of the upper and lower surfaces, the photography process is quite time-consuming. For the classic HiSeq 2000, it takes about 40 minutes for the fluorescence signal generated by one sequencing cycle to be collected by taking photos, which is longer than the sequencing reaction itself.

3 Cutting

After collecting the fluorescence signal, turn off the laser and use a cutting agent to remove the fluorescent and terminating groups marked by the bases on the extension products of the sequencing primers. Both modifying groups have been removed, which eliminates interference with fluorescence and restores the base at the end of the chain to its natural and extensible state, preparing for the next round of sequencing.

Repeat the steps of synthesis, excitation, and collection to sequence the second base.

The sequencing cycle can be repeated multiple times. In theory, it can be repeated until the signal-to-noise ratio decreases to the point where the base signal cannot be effectively recognized. Due to the continuous decay of the luminescence efficiency of fluorescent groups and the activity of Taq enzyme, the number of sequencing cycles is actually limited, and the large number of repetitions is related to the formulation of SBS reagent. The number of sequencing cycles can be manually set during the software setup process, and this repetition is the read length of each sequencing fragment. At present, the Illumina platform has various read lengths such as 2x100 bp, 2x125 bp, 2x150 bp, 2x300 bp, among which 2x150 bp is widely used.

Single read sequencing: First, the DNA sample is fragmented to form a 200-500 bp fragment. Primer sequences are connected to one end of the DNA fragment, and then a linker is added to the end to fix the fragment on a flow cell to generate a DNA cluster. The sequence is then read at the single end by machine sequencing.

Paired end sequencing: The paired end sequencing method refers to adding sequencing primer binding sites to both ends of the adapter when constructing the DNA library to be tested. After the completion of the * round sequencing, the template strand of the * round sequencing is removed, and the paired end module is used to guide the complementary strand to regenerate and amplify at its original position, in order to achieve the template amount used for the second round sequencing, and then perform the synthesis sequencing of the second round complementary strand.

A genomic library refers to a collection formed by cutting all the genomic DNA of a certain organism into DNA fragments of a certain length and cloning them onto a certain vector. Genomic libraries are divided into nuclear genomic libraries, chloroplast genomic libraries, and mitochondrial genomic libraries based on their DNA sources. When constructing a genomic library, the genetic information donor is genomic DNA, so there is no developmental stage or tissue organ specificity. A genomic library contains clones of all coding and non coding sequences on genomic DNA; Every gene of a biological organism has its clone in the library, and the cloned gene fragments include spacer sequences, so the genome library can truly display all the structural information of the genome.

Genomic Library Definition: The complete genetic information of a certain organism's genome is stored in a recipient bacterial clone population through a cloning vector, and this population is the genome library of this organism.

20024904 Illumina Sequencing Kit v2.5 in stock discount

Product Name: Illumina 20024904 NextSeq 500/550 v2.5 Sequencing Kit (150 cycles)

Product Code: 20024904

Product brand: Illumina/Imena USA

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Illumina NextSeq 500/550 v2.5 Sequencing KitSpot supply, welcome to inquire