[pmiguel]

Hi, I think i got my answer, thanks. If you can also tell me in Illumina sequencing which sequencing read length format generates overlapping end? and how long is the overlapping region for 230bp insert?
Thank you so much

I am sure you can do the calculation yourself.

--
Phillip

[bssharma]

I am sure you can do the calculation yourself.

--
Phillip

According to my calculation there will not be any overlapping. Plz correct me if i am wrong.
Thanks!

[pmiguel]

According to my calculation there will not be any overlapping. Plz correct me if i am wrong.
Thanks!

That would depend on the length of the reads.

--
Phillip

[jngao1204]

Thanks for sharing!

[shirley971002]

PCR primers sequences?

The 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.

sorry,I am new.I want to know, in Truseq vs old adapters.pdf, which are PCR primers? Can you give the specific sequences? Thank you !

[ramkrissh]

Can any body has details of illumina miseq sequencing for 16S amplicon library construction specific to V3 region only . I need primer , adapter and barcode sequences used for V3 region 16S sequencing ..... Please help

[Katerina Cvikova]

Hi everybody,
do you know if it is possible to sequence in one run on MiSeq libraries from Nextera DNA and TruSeq PCR-Free together?

[nucacidhunter]

Yes. They are perfect match if prepared by standard method. Both would have relatively similar peak size, so preferential clustering of smaller fragments will not be an issue and you can expect to get reads from both in proportion to input library.

[Sarutis]

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

[relipmoc]

Skewer supports demultiplexing amplicon pairs

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

Recently we indexed 6 amplicon libraries and sequenced them in a 2 x 300bp MiSeq run. Each of these 6 amplicon libraries was also a mixed library which contains about 60 samples barcoded by different combination of forward-primer sequence and reverse-primer sequence (In fact, fragments from these samples were also ligated with 6 nucleotides index sequences). Hence we may demultiplex about 360 samples using this protocol.

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.

[Sarutis]

Thanks, relipmoc
Since you were so kind to answer quickly, I'll ask few more things:
what do you use to connect overlapping paired sequences? I found PEAR and stitch as avaliable softwares, but I'd like an expert opinion

[ssully]

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

versus this set?

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)

[ssully]

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:

universal                                                                                                                indexed
5'             AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert--AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3'
                                                                |||||||||||||||||||||||||||||||||||||                          
3'     GTTCGTCTTCTGCCGTATGCTCTNNNNNNAGCACTACACTGACCTCAAGTCTGCACACGAGAAGGCTAGA---insert--TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA  5'
indexed                                                                                                                  universal

If there is a PCR enrichment step (which is omitted from the new PCR-free kits) the PCR primers are

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:

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.

[ksil91]

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

[Ingeneious]

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:

universal                                                                                                                indexed
5'             AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert--AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3'
                                                                |||||||||||||||||||||||||||||||||||||                          
3'     GTTCGTCTTCTGCCGTATGCTCTAGNNNNNNCACTACACTGACCTCAAGTCTGCACACGAGAAGGCTAGA---insert--TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA  5'
indexed                                                                                                                  universal

I think your index in the 3' to 5' read was off for so I adjusted it. Based on the 07-14 Illumina Customer letter for TruSeq HT indexes, the dual index would look something like this?

5' AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATC-T-insert-A-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7]ATCTCGTATGCCGTCTTCTGCTTG 3'
			      	                      |||||||||||||||||||||||||||||||||||
3'     GTTCGTCTTCTGCCGTATGCTCTA[i7]CACTGACCTCAAGTCTGCACACGAGAAGGCTAG-A-insert-T-CTAGCCTTCTCGCAGCACATCCCTTTCTCACA[i5]CACATCTAGAGCCACCAGCGGCATAGTAA 5'