What is a paired-end library?
Paired-end sequencing allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data. Paired-end sequencing facilitates detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.
What is library construction in NGS?
Fundamental to NGS library construction is the preparation of the nucleic acid target, RNA or DNA, into a form that is compatible with the sequencing system to be used (Figure 1).
What happens during the library preparation phase of the Illumina system?
Library preparation is crucial to the success of your NGS workflow. This step prepares DNA or RNA samples to be compatible with a sequencer. In the Illumina sequencing workflow, these adapters contain complementary sequences that allow the DNA fragments to bind to the flow cell.
How many files are generated in a paired-end Illumina sequencing?
We use the Illumina MiSeq platform, which we find typically generates 8–10 million paired-end reads per sequencing run.
How does paired end sequencing work in Illumina?
Illumina Paired End Sequencing Illumina gets sequence data from both strands of input sequence which means it outputs data from both ends of the input and is normally reported two files R1 and R2, often refereed to as mates files (R1=first mates, R2=second mates).
How are data files reported in Illumina paired end information?
Illumina gets sequence data from both strands of input sequence which means it outputs data from both ends of the input and is normally reported two files R1 and R2, often refereed to as mates files (R1=first mates, R2=second mates). Due to the way data is reported in these files, special care has to be taken when processing these data files.
What kind of DNA is used for Illumina library construction?
Starting material for Illumina library construction is usually double stranded (ds) DNA from any source: genomic DNA, BACs, PCR amplicons, ChIP samples, any type of RNA turned into ds cDNA (mRNA, normalized total RNA, smRNAs), etc. Pretty much anything you can think of that ends up as, or can be turned into, dsDNA.
How are index adapters used in Illumina Library prep?
Illumina Index Adapters Explained Nextera library prep uses an engineered transposome to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in a single step. A limited-cycle PCR step uses the adapters to amplify the insert DNA.
