Ligated sequences
• Adapters
• Primers
• Tags
• Barcodes
• UMIs
• Spacers
• Linkers
Why to ligate anything?
1) Bind to a flow cell for next generationsequencing
2) Allow for PCR enrichment of adapter‐ligatedDNA fragments only
3) Allow for indexing or “barcoding” of samples so multiple DNA libraries can be mixed together
into 1 sequencing lane (known as multiplexing)
4) Error discovery with tags
Adapters
Ligated sequences
Have to be present:
P5/P7 – adaptersfor flowcell binding
SP1/SP2 – sequencing primer bindingsite
Optional – but often used
i5/i7 – Sample index – to recognize sequenced libraries
Optional:
Barcode – uniquefor sample, cell
UMI – Unique MolecularIdentifier – to identifytechnical duplicates
Ligated sequences
Spacers
- If combiningdifferent lengthslibraries
Linkers
- for better merging of sequences
Demultiplexing
Sorting reads according to sample. Based on index/barcode.
Forward barcode: AGGCT
Reverse barcode: CAATG
3‘ – T C C G A A T G G C C ............... A T C C G G T A A C – 5‘
5‘ – A G G C T T A C C G G ............... T A G G C C A T T G – 3‘
>read_1
AGGCTATTTAGCGCTACGTAATTTAGCCAATG
>read_2 *
AGGCTATTTAATTTAATATTTAGCATTTAGCCATTG
>read_3
CATTGATTTAGCATTTAGCATATTTAGCAAGCCT
> read_4
AGGCTATATTTATTTAGCATATTTAGCATTCAATG
> read_5
CAATGATTTAGCATTTAGCATTTAGCATTAGGCT
> read_6
CAATGATTTAGCATATTTAATTTAGCATAGGCT
> read_7
AGCCTATTTAGCATTATTTAGCATTTAGCCATTG
> read_8 *
CAATGATTTAATTTAGCATATTTAGCAGCCT
UMI
*https://www.lexogen.com/rna-lexicon-what-are-unique-molecular-identifiers-umis-and-why-do-we-need-them/
UMI
*https://www.lexogen.com/rna-lexicon-what-are-unique-molecular-identifiers-umis-and-why-do-we-need-them/
UMI
*https://www.lexogen.com/rna-lexicon-what-are-unique-molecular-identifiers-umis-and-why-do-we-need-them/
Linker
Linker
Adapters