The nature of Illumina sequencing is still sequencing by synthesis (SBS). Any SBS method requires the presence of adaptors, which are short double-stranded DNA oligos whose sequences are known to us. The adaptors are designed by scientists, and there are a few popular adaptor sequences that are used in the NGS field. The following three are the most popular ones:

5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT -insert-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 Truseq Read 1 Truseq Read 2 i7 Illumina P7

5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN ACACTCTTTCCCTACACGACGCTCTTCCGATCT -insert-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 i5 Truseq Read 1 Truseq Read 2 i7 Illumina P7

5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG -insert-CTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC -insert-GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 i5 Next era Read 1 Next era Read 2 i7 Illumina P7

There are some other adaptors that can be used, but they either become obsolete or not used usually. You can check the Illumina adapter sequences document for more details. Basically, the nature of an Illumina library preparation is the process of adding those coloured adaptor seuqences to both sides of the DNA of your interest, which is the "-insert-" bit in the above examples. All the commercial kits you buy for the library preparation do exactly that.

How to add those adaptors? Well, this is where you can become creative. In this GitHub repository, many single cell genomics methods are shown. They all do one thing: add those adaptors to the sides of the DNA they want to sequence. The "N"s in the above examples are the indices, or barcodes that discriminate different samples. The index at the right hand side is often called "i7", or index1; the index at the left hand side is called "i5", or index2. This is because the index "i7" is sequenced first before "i5" is sequenced.

The only required seqeuences are the following two:

Illumina P5 adaptor: 5'-

AATGATACGGCGACCACCGAGATCTACAC -3'Illumina P7 adaptor: 5'-

CAAGCAGAAGACGGCATACGAGAT -3'

If you use Illumina sequencing, you have to use and make sure they are at the sides of your DNA fragments, like shown in the three examples above. The adaptor sequences in the middle like

Once those adaptors are added propertly, we are ready to sequence them using Illumina machines. In the sequencing reagents provided by Illumina, the seuqencing primers are actually a mixture of different primers, including Truseq, Nextera and even those primers from kits that are obsolete. Therefore, you actually can sequence different types of libraries together. For example, Truseq librareis and Nextera libraries can be mixed together and sequenced together without any problem. There are some slight differences among different Illumina machines, in terms of how they sequence Read 1, Read 2, Index 1 and Index 2. Check this document for more details.

Truseq Single Index Library: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT ----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Truseq Dual Index Library: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT ----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Nextera Dual Index Library: 5'- TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG ------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC -insert-GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Truseq Single Index Library: 5'- AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Truseq Dual Index Library: 5'- AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Nextera Dual Index Library: 5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC -insert-GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Truseq Single Index Library (not really needed): 5'- AATGATACGGCGACCACCGAGATCTACAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Truseq Dual Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Nextera Dual Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC -------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC -insert-GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5'

Truseq Single Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT -insert-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' <-------TGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA -5'

Truseq Dual Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN ACACTCTTTCCCTACACGACGCTCTTCCGATCT -insert-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' <-------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA -5'

Nextera Dual Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG -insert-CTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' <-------AGCAGCCGTCGCAG TCTACACATATTCTCTGTC -5'

Truseq Single Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT -insert-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' <------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'

Truseq Dual Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN ACACTCTTTCCCTACACGACGCTCTTCCGATCT -insert-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' <------TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG -5'

Nextera Dual Index Library: 5'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG -insert-CTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3' <------GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'