Library preparation of tagged nucleic acids utilizing a single-tube addition protocol
A tagging and tagging technology, applied in the field of preparing nucleic acid fragment libraries, can solve problems such as the inability to ensure the representativeness of target nucleic acid and the loss of target nucleic acid.
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Embodiment 2
[0092] Example 2 illustrates a protocol for the labeling step using the methods provided herein. In embodiments in which a single cell is used to prepare a library for sequencing, there are only two copies of the genome, and therefore a smaller insert size tends to increase library diversity. As shown in Example 8, the count increases as the insert size decreases, and therefore the diversity represented by the library increases as the insert size decreases. Therefore, in some embodiments, the methods herein use higher amounts of transposase in the tagging step to increase fragmentation and reduce the insertion size of the tagged nucleic acid fragments. As shown, when 1 μl Tn5 is used in the tagging reaction, the average fragment size is about 550 bp; and when 2 μl Tn5 is used in the tagging reaction, the average fragment size is about 400 bp. Consistent with the smaller insert size, the diversity of the library when treated with 2μl of Tn5 was increased compared to the diversi...
Embodiment 3
[0111] Example 3 illustrates the limited cycle PCR amplification that can add other sequences at both ends of the tagged nucleic acid fragments, such as indexer 1 (i7) and indexer 2 (i5) (from Illumina, Inc. , San Diego, CA) and sequences required for other purposes such as cluster formation. In single-cell sequencing, the input DNA is relatively small, and therefore the number of PCR cycles can be adjusted to achieve better sequencing results. In Example 9, a single cell was used as the starting material to test and optimize the number of PCR cycles. As shown, when PCR with 16 cycles is used in copy number analysis, the noise is large, and when PCR with 18 cycles or 20 cycles is used, the noise is significantly reduced. Therefore, in some embodiments, the number of PCR cycles is 18, 19, or 20.
[0112] For the amplification reaction by PCR known to those skilled in the art, many kinds of enzymes and kits are available. For example, in some embodiments, FAILSAFE from EPICENTRE...
Embodiment 11
[0129] Example 11 compares this method with some current single cell preparation methods. When using the REPLI-g single-cell kit developed by QIAGEN (SanDiego, CA) to prepare nucleic acids, the copy number variation data is noisy when derived from a single cell, three cells, or five cells. When SurePlex (PicoPlex) developed by Illumina, Inc (San Diego, CA) is used to prepare nucleic acids, it reduces noise compared to the REPLI-g single cell kit. As shown, this method (Nextera SC) further reduces noise compared to using the SurePlex amplification system. Therefore, this method provides an advanced method for analyzing copy number variation.
[0130] One aspect of copy number variation analysis is to detect mosaicism. Mosaic or mosaic phenomenon indicates the existence of two or more genotypes in an individual. There are two types of mosaic phenomena: somatic mosaicism and germline mosaicism. Somatic cell mosaicism occurs when a somatic cell contains more than one genotype due...
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