For example, you can use Truseq Read 1 at the left hand side, and Nextera Read 2 at the right hand side. In addition, you can also mix different types of adaptors due to the reaons mentioned in the "Library sequencing" section below. All the commercial kits you buy for the library preparation do EXACTLY that, no matter where your buy them, no matter what you want to sequence. 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. If you are interested, you can check the Illumina adapter sequences document for full details. There are some other adaptors that can be used, but they either become obsolete or not used usually. Illumina P5 i5 Next era Read 1 Next era Read 2 i7 Illumina P7 Illumina P5 i5 Truseq Read 1 Truseq Read 2 i7 Illumina P7 Nextera Dual Index Library:ĥ'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG-insert- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3'ģ'- TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN AGCAGCCGTCGCAG TCTACACATATTCTCTGTC-insert- GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5' Illumina P5 Truseq Read 1 Truseq Read 2 i7 Illumina P7 Truseq Dual Index Library:ĥ'- AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN ACACTCTTTCCCTACACGACGCTCTTCCGATCT-insert- AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3'ģ'- TTACTATGCCGCTGGTGGCTCTAGATGTG NNNNNNNN TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-insert- TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5' The following three (actually two main types to be honest) are the most popular ones:ĥ'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTCCGATCT-insert- AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNNNN ATCTCGTATGCCGTCTTCTGCTTG -3'ģ'- TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAGGCTAGA-insert- TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5' The adaptors are designed by scientists, and there are a few popular adaptor sequences that are used in the NGS field. Any SBS method requires the presence of adaptors, which are short double-stranded DNA oligos whose sequences are known to us. The nature of Illumina sequencing is still sequencing by synthesis (SBS). (3) Cluster regeneration, add Read 2 sequencing primer to sequence the second read (top strand as template, sequence cDNA):ĥ'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGG AAGCAGTGGTATCAACGCAGAGTACGT NNNNNNNNNNNN NNNNNNNN(dT)XXX.Illumina sequencing About Illumina sequencing libraries (2) Add i7 index sequencing primer to sequence the i7 index (bottom strand as template):ĥ'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC-> |-5'- TTTTTTTT AAGCAGTGGTATCAACGCAGAGTACGT (dT)XXX.XXX CTGTCTCTTATACACATCTĪAAAAAAA TTCGTCACCATAGTTGCGTCTCATGCA (pA)XXX.XXXXXXXXXXXX GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTG -5'ģ'- TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCC TTCGTCACCATAGTTGCGTCTCATGCA NNNNNNNNNNNN NNNNNNNN(pA)XXX.XXX GACAGAGAATATGTGTAGA GGCTCGGGTGCTCTGNNNNNNNN TAGAGCATACGGCAGAAGACGAAC -5' TTCGTCACCATAGTTGCGTCTCACTTACCCXXXXX.XXXXX(dT) TGCA TGAGACGCAACTATGGTGACGAATTTTTTTT -5'-| Step-by-step library generation (1) mRNA capture using Beads-oligo-dT in the droplets:ĥ'- AAGCAGTGGTATCAACGCAGAGTGAATGGGXXXXX.XXXXX(pA) ACGT ACTCTGCGTTGATACCACTGCTTAAAAAAAA I7 index sequencing primer: 5'- CTGTCTCTTATACACATCT CCGAGCCCACGAGAC -3' Read 2 sequencing primer: 5'- GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG -3' Read 1 sequencing primer (seqB): 5'- GCCTGTCCGCGG AAGCAGTGGTATCAACGCAGAGTAC -3' Read 1 sequencing primer (seqA): 5'- GCCTGTCCGCGG AAGCAGTGGTATCAACGCAGAGTACGT -3' Library PCR primer 2 (this is basically Nextera N7xx): 5'- CAAGCAGAAGACGGCATACGAGAT GTCTCGTGGGCTCGG -3' Library PCR primer 1: 5'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGG AAGCAGTGGTATCAACGCAGAGTAC -3' Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3' Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3' Nextera N7xx primer entry point (s7): 5'- GTCTCGTGGGCTCGG -3' ![]() Nextera N/S5xx primer entry point (s5): 5'- TCGTCGGCAGCGTC -3' Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'- AGATGTGTATAAGAGACAG -3' Template Switching Oligo (TSO): 5'- AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG -3' Seq-Well used exact the same oligo design with Drop-seq with Beads-oligo-dT-seqB, which was published in Nature Methods 14, 395–398 (2017).īeads-oligo-dT-seqA: |-5'- TTTTTTT AAGCAGTGGTATCAACGCAGAGTACGT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3'īeads-oligo-dT-seqB: |-5'- TTTTTTT AAGCAGTGGTATCAACGCAGAGTAC TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT -3' Here, Beads-oligo-dT-seqA was used as demonstration. In the original pulication in Cell 161, 1202-1214 (2015), there are two batches of beads, with only two base pairs difference.
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