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PCR primers sequences?
Originally posted by protist View PostThe original adapters (what I called legacy) released by Illumina are not exactly the same sequence as the currently in use TruSeq adapters - e.g. old adapters did not have indexes TruSeq ones do. The sequences for the individual TruSeq adapters can be found in the Illumina customer letter (see attached) which can also be downloaded from the Illumina website. I have also attached an alignment I had illustrating the differences between the original adapters and the currently in use TruSeq adapters.
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2x300 MiSeq amplicon libraries barcoded primers
I'm new to MiSeq sequencing and to this blog and I'm having difficulties:
I'm sure these info are somewhere in the blog, but would someone be so kind and tell me where to find the primer design info for barcoded amplicon library for a 2x300 MiSeq run?
Are there any other info needed
Thanks in advance
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Skewer supports demultiplexing amplicon pairs
Originally posted by Sarutis View PostI'm new to MiSeq sequencing and to this blog and I'm having difficulties:
I'm sure these info are somewhere in the blog, but would someone be so kind and tell me where to find the primer design info for barcoded amplicon library for a 2x300 MiSeq run?
Are there any other info needed
Thanks in advance
In my understanding, the first level barcoding is easy to handle, you should note down the 6 bp index sequences when construct the libraries, and later you can specify the 6 bp sequences in the Sample Sheet when preparing a MiSeq run. For the second level barcoding, you need to know what gene region is amplified with what primers (forward and reverse).
For demultiplexing, I suggest you use skewer (http://sourceforge.net/projects/skewer/) which supports demultiplexing amplicon pairs.
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I'm quite lost. I've got a set of paired end 100 nt HiSeq reads (library insert size was ~300bp) from a colleague and I can't determine what adapter sequences to trim. The current Illumina letter to customers lists a variety of sequences. Can someone explain the use of this set
Oligonucleotide sequences for Paired End DNA
PE Adapters
5' P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
PE PCR Primer 1.0
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
PE PCR Primer 2.0
5' CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
PE Read 1 Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
PE Read 2 Sequencing Primer
5' CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
Oligonucleotide sequences for the Multiplexing Sample Prep Oligo Only Kit
Multiplexing Adapters
5' P-GATCGGAAGAGCACACGTCT
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Multiplexing PCR Primer 1.0
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
Multiplexing PCR Primer 2.0
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
Multiplexing Read 1 Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Multiplexing Index Read Sequencing Primer
5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
Multiplexing Read 2 Sequencing Primer
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
PCR Primer, Index 1
5’ CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTC
PCR Primer, Index 2
5’ CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTC
.
.
.etc for 12 indexes total..
For a paired end set that includes indices (barcodes) which set would have been used?
Also, what are the 2 sequences of the oligos that coat a paired end flowcell used in the current HiSeq platform? (these would be sequences complementary to adapters sequences)Last edited by ssully; 11-17-2014, 07:22 PM.
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Illumina helped sort me out. The seqs I posted last time are from an old , discontinued version of the kit (pp 20-21 of the July 2014 letter). The newer TruSeq adapters('universal' and 'indexed') are found on pp 12-13 of the letter .
These 'floppy end' adapters dimerize only at their last 12 nt. After they're ligated to flanks of the insert DNA fragment the result looks like this:
Code:universal indexed 5' AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert--AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3' ||||||||||||||||||||||||||||||||||||| 3' GTTCGTCTTCTGCCGTATGCTCTNNNNNNAGCACTACACTGACCTCAAGTCTGCACACGAGAAGGCTAGA---insert--TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5' indexed universal
P1: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTA
(i.e. the first 44 bases of the universal adapter)
P2: 5' CAAGCAGAAGACGGCATACGAGAT
(reverse complement of the last 24 bases of the indexed primer)
P2 primes first, and then the ssDNA from P2 priming/extension becomes the template for P1. So the PCR product is:
Code:5'AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert-----AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3' 3'TTACTATGCCGCTGGTGGCTCTAGATGTGTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA---insert-----TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC 5'
The enriched PCR product can then be denatured and each strand hydbridized to the bound flowcell oligos for cluster generation and reading.Last edited by ssully; 11-18-2014, 01:57 PM.
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Indexed PCR Primer Ordering
Hello, I'm new here so apologies if this question has been answered.
I'm following the Buckler Lab GBS protocol and only have access to 48 adapters for ApeKI. I'd like to multiplex these into one Illumina lane using indexed PCR primers. I found sequences for PCR primers from the ddRAD protocol(Peterson). Do I have to alter the sequence at all to match my ApeKI adapters? Do I have to request anything particular when ordering these primers from IDT? Or would it be better for me to just order the NEBNext Multiplex Oligos for Illumina Kit? (https://www.neb.com/products/e7335-n...-primers-set-1)
ApeKI Common Adapter:
5'-CWGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
5'-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
ApeKI Barcoded Adapter:
5’-CWGxxxxAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTyyyy
PCR primers I'm considering ordering:
PCR1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
PCR2_Idx_1_ATCACG: CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_2_CGATGT: CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_3_TTAGGC: CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_4_TGACCA: CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGC
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Originally posted by ssully View PostIllumina helped sort me out. The seqs I posted last time are from an old , discontinued version of the kit (pp 20-21 of the July 2014 letter). The newer TruSeq adapters('universal' and 'indexed') are found on pp 12-13 of the letter .
These 'floppy end' adapters dimerize only at their last 12 nt. After they're ligated to flanks of the insert DNA fragment the result looks like this:
Code:universal indexed 5' AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert--AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3' ||||||||||||||||||||||||||||||||||||| 3' GTTCGTCTTCTGCCGTATGCTCTAGNNNNNNCACTACACTGACCTCAAGTCTGCACACGAGAAGGCTAGA---insert--TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5' indexed universal
Code:5' AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATC-T-insert-A-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7]ATCTCGTATGCCGTCTTCTGCTTG 3' ||||||||||||||||||||||||||||||||||| 3' GTTCGTCTTCTGCCGTATGCTCTA[i7]CACTGACCTCAAGTCTGCACACGAGAAGGCTAG-A-insert-T-CTAGCCTTCTCGCAGCACATCCCTTTCTCACA[i5]CACATCTAGAGCCACCAGCGGCATAGTAA 5'
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