Illumina library preparation..........

Illumina library preparation.........

Input amounts as low as 5ng; high fidelity amplification with minimized GC bias; fast workflow (<3 hours) 

Fragmentation of DNA ; repair of 3’ and 5’ ends to form blunt-ended, phosphorylated molecules, and the addition of a non-templated dA-tail before ligation to an adaptor; final step is PCR amplification of the library (PCR step is used if sufficient yield is required).

(https://www.neb.com/applications/library-preparation-for-next-generation-sequencing/illumina-library-preparation/dna-for-illumina)

Kits are available for the sample preparation (eg. NEBNext® kits). Different kits for different purpose. (genomic DNA, ChIP DNA, FFPE DNA). Kits include fragmentation reagent, end repair and dA-tailing reagents, 

#If reagents are in the same tube, cleanup  is  not needed, so sample is not lost. Reagents have been validated by preparation of library from a reference sample, followed by sequencing.

Use with DNA in standard buffers (TE, Tris-HCl) and water 

Vary incubation time to generate a wide range of insert sizes 

Kits can combine shearing, end-repair, dA-tailing, and linker-ligation

mRNA libraries are prepared by construction of a cDNA library, followed by DNA library preparation steps of repair of 3′ and 5′ ends;  addition of a non-templated dA-tail before ligation with an adaptor.; libraries are size selected after adaptor ligation, and amplified via PCR

Small RNA libraries are constructed using a different workflow, in which adaptors are ligated directly to the small RNA molecules, followed by reverse transcription, PCR amplification and size selection. 

(http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026969)

TruSeq DNA Nano with Single Indexes (supports 24-sample manual processing for low-throughput (LT) studies). TruSeq DNA Nano with 96 CD Indexes  supports 96-sample processing for high-throughput (HT) studies.

(https://www.abmgood.com/marketing/knowledge_base/next_generation_sequencing_experimental_design.php)

VeriSeq PGS Kit-MiSeq can give screening of all 24 chromosomes for selection of euploid embryos. Cells used in this system can come from various stages of embryo development: polar body 1, polar body 2, blastomere biopsy from day 3 embryos, and trophectodermal (TE) biopsy from blastocysts. Single cell samples simulate blastomere biopsies and three-cell samples to simulate trophectoderm biopsies. Sample preparation done with SurePlex DNA Amplification Kit. Samples sequenced per Run is 24. PGS  results from VeriSeq PGS (synthesis (SBS) chemistry) are comparable to that of array-based 24sure technology. Analysis software isis BlueFuse Multi v4.1

(https://www.nggthailand.com/preimplantation-genetic-diagnosis-pgd/)

(https://www.slideshare.net/Research_Instruments/new-solutions-for-genetic-testing-of-embryos)

The reversible terminator–based method enables massively parallel sequencing of millions of DNA fragments, detecting single bases as they are incorporated into growing DNA strands. This method

lowers sequencing-related errors. High data quality results in low false positive and false negative rates, reducing the need for extensive downstream validation while providing full confidence in the data. About 15 Gb of data with 25M sequencing reads is produced.

The VeriSeq PGS steps (takes about 12 hours (sample to report, with robust, low-noise results).

 DNA extraction from  a single embryo cell ..... whole-genome amplification (WGA) using

the SurePlex™ DNA Amplification Kit.......amplified samples

* Q30 = 1 error in 1000 base calls or an accuracy of 99.9%.

Prepared libraries are loaded onto a flow cell for sequencing on the MiSeq System.

An on-instrument computer performs data analysis. Generated files are then imported into BlueFuse

Multi Software for analysis, data management, and results reporting.

(https://www.nggthailand.com/preimplantation-genetic-diagnosis-pgd/)

#Reduce library bias and coverage gaps. GC-rich regions, promoters, and repetitive regions offer poor coverage. Chaotic and mosaic samples were excluded from analysis.