How does the adapter in Illumina sequencing work?

Why are adapter sequences trimmed from only the 3' ends of reads? https://support.illumina.com.cn/bulletins/2016/04/adapter-trimming-why-are-adapter-sequences-trimmed-from-only-the--ends-of-reads.html

Expands one nucleic base at a time https://www.researchgate.net/figure/Illumina-sequencing-process-A-DNA-library-Breaks-the-genome-DNA-to-form-DNA_fig3_357155980

In Illumina sequencing, the barcode (also known as the index) is indeed a critical part of the sequencing process because it allows for the identification and demultiplexing of multiple samples that are sequenced together in the same run.

Here’s how it works:

• Adapter Ligation: First, adapters are ligated to the fragmented DNA. These adapters contain the sequences for P5 and P7 priming sites, necessary for flow cell attachment and the initiation of the sequencing reaction.

• Index Sequences: The adapters also include index sequences (barcodes). In the case of dual-indexing, one index (Index 1) is on the adapter ligated at the P7 end, and another index (Index 2) is on the adapter ligated at the P5 end. These indexes are unique to each sample.

• Sequencing Initiation: Sequencing begins with the binding of sequencing primers to their complementary sites on the adapters—not directly from the index sequences. However, the index sequences are read during specific additional sequencing reactions:

  * For Read 1, sequencing starts from the P5 end.
  * If performing paired-end sequencing, after Read 1 is complete, the machine performs a read of the Index 1 sequence.
  * Then, the flow cell is reconfigured to sequence Read 2 from the P7 end.
  * Finally, if dual-indexing, the Index 2 sequence is read.
  

• Index Reading (Read1 Primer and i7 Index Primer): The indexes are not part of the main sequence reads (Read 1 or Read 2) but are read in separate, dedicated sequencing reactions using specific index primers after the completion of the standard sequencing cycles.

The crucial point is that the sequencing of the index sequences happens after the main DNA fragment has been sequenced, during dedicated index read cycles. The readout of the indexes is integral to the sequencing run and allows the software to assign each sequence to the correct sample in the analysis phase, enabling the pooling of multiple samples in a single sequencing run. This process is called demultiplexing.

Tn5 adapter https://teichlab.github.io/scg_lib_structs/methods_html/plate_and_piATAC-seq.html

Y-shaped-adaptors https://www.researchgate.net/figure/DNA-template-ligation-with-Y-shaped-adaptors-Blunt-ended-ds-DNA-templates-5_fig2_323640739

    (0) Final library structure:
    5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
        TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
            Illumina P5              i5         s5              ME                cDNA                ME               s7          i7            Illumina P7


    Library sequencing:
    (1) Add read 1 sequencing primer to sequence the first read (bottom strand as template):

                                                                Primer1
                                        5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG|--READ1---->
    3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

    (2) Add index 1 sequencing primer to sequence the first index (i7) (bottom strand as template, 8 cycles):


                                                                                            5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------>
    3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNAGCAGCCGTCGCAGTCTACACATATTCTCTGTCXXXXXXXX...XXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

    (3) Cluster regeneration, add Index 2 sequencing primer to sequence the second index (i5) (top strand as template, 8 cycles. Single cells can be identified as the combination of i5 and i7):


    5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                    <-------AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5'

    (4) Add read 2 sequencing primer to sequence the second read (top strand as template):


    5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXX...XXXXXXXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
                                                                                    <----READ2--|GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                        Primer2

like unlike

点赞本文的读者

还没有人对此文章表态

本文有评论

没有评论

看文章，发评论，不要沉默