Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • VC87
    replied
    Thanks Brian, i will definitely have a look at BBDuk!

    Leave a comment:


  • Brian Bushnell
    replied
    I think the easiest thing to do would be to filter out low-quality reads and then run FastQC on the high-quality reads to see if they have any problems. This is a bit tangential to the topic, but for example you can remove low-quality reads with BBDuk using the maq (min average quality) flag, like "maq=15".

    On a more thread-related note, the BBMap package has all of the Illumina adapter sequences in a fasta file which is more convenient than Illumina's PDF format. It's in /bbmap/resources/adapters.fa once you unzip/untar the package. As pmiguel indicated, this is just adapters; there are a lot of other sequences that Illumina considers proprietary and forbids sharing.

    I'd also like to note that if you have paired-end reads with an unknown adapter sequence, you can discover it with BBMerge like this:

    bbmerge.sh in1=r1.fq in2=r2.fq outa=adapters.fa

    This tends to be useful in situations like discovering PhiX adapters, which are not in Illumina's customer letter.
    Last edited by Brian Bushnell; 05-28-2016, 07:57 AM.

    Leave a comment:


  • VC87
    replied
    Hi everyone!
    Does anyone knows if/how is ir possible to distinguish kmer plots in FASTQC of reads derived from RRBS librarys from reads that just have bad quality?thanks in advance

    Leave a comment:


  • HESmith
    replied
    The document (yes, you have the latest version) lists all of the adapter sequences ever offered by Illumina (or Nextera, whose technology it acquired). The methods have evolved, from single-end to paired-end sequencing, and unindexed to single indexing to dual indexing. All of those changes have required changes in the adapter sequences (and sequencing/indexing primers). And Illumina does offer a separate document that indicates which of their library prep versions are or are not compatible with which sequencing kits/versions.

    Publications are a lagging indicator, so the sequences in a paper may be obsolescent by the time of release.

    I explicitly stated which adapters are compatible with the current version of sequencing. Your service provider should be able to verify this information for you, and/or assist you with primer design.

    Leave a comment:


  • ajthomas
    replied
    Originally posted by HESmith View Post
    The sequences that you list are from the earliest version of Illumina multiplexing, which was supplanted by the TruSeq platform several years ago. If you want your adapter sequences to be compatible with the current generation of sequencing and index primers, download the Customer Sequence Letter from Illumina and use the TruSeq v1/v2 (pp. 12-13) Universal Adapter plus the reverse complement of the Truseq Indexed Adapter (plus 3' A) to design your amplicon primers. If you require dual indexing, use the TruSeq HT sequences (D500 + D700 rev comp, pp. 10-11) as the basis of your design.
    I got those sequences from the copy of the letter that I downloaded about a month ago. It is dated August 2014 and is the current version available from Illumina today. If those are old sequences that are no longer supported, Illumina needs to make it clear in the letter that they are obsolete and included only for legacy reasons.

    The sequences I listed that are labeled for multiplexing are the same as those listed for TruSeq, so I've been tempted to go with those. However, I can't be sure because there are so many different sequences out there. Both the Nextera and small RNA sample prep kits use still different sequences. Furthermore, a recent publication (Nature Methods, published last year) from which I and some colleagues are trying to replicate some methods used oligos that match the PE sequences listed earlier, not the TruSeq sequences. It seems that Illumina is still using a few different adapter sequences, and I don't understand how that can be.

    Leave a comment:


  • HESmith
    replied
    Originally posted by ajthomas View Post
    Which is the correct sequence?
    The sequences that you list are from the earliest version of Illumina multiplexing, which was supplanted by the TruSeq platform several years ago. If you want your adapter sequences to be compatible with the current generation of sequencing and index primers, download the Customer Sequence Letter from Illumina and use the TruSeq v1/v2 (pp. 12-13) Universal Adapter plus the reverse complement of the Truseq Indexed Adapter (plus 3' A) to design your amplicon primers. If you require dual indexing, use the TruSeq HT sequences (D500 + D700 rev comp, pp. 10-11) as the basis of your design.

    Leave a comment:


  • ajthomas
    replied
    Originally posted by ssully View Post
    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



    versus this set?



    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)
    I've been trying to sort this out to order some primers to use for making amplicon libraries on the Fluidigm Access Array. The Fluidigm protocol uses non-standard sequences (and therefore custom sequencing primers), and is not compatible with PE sequencing. I want to modify it to use standard Illumina sequences so that the libraries are PE compatible and fit better into the Illumina ecosystem.

    The confusion comes from these adapter sequences:
    PE PCR Primer 2.0
    5' CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
    PE Read 2 Sequencing Primer
    5' CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT

    Multiplexing PCR Primer 2.0
    5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
    Multiplexing Read 2 Sequencing Primer
    5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
    The sequence of the adapter used to attach the i7 adapter (and index) is different. Either one should work to make a library, but the problem will come when sequencing read 2 and reading the first index. A different sequencing adapter will be needed. Which is the correct sequence?

    Leave a comment:


  • troubleble
    replied
    (reply to clueheart)

    Just wanna remind you:
    HP11 combines at least 2 kinds of sequencing primer.
    The sequence you posted should be one of them, but I dont know the exact letter too, maybe no one knows except Illumina.
    btw, the other primer should fit NextEra system.

    Leave a comment:


  • enum
    replied
    Hi,

    I'm following the protocol from this TCR sequencing paper: http://www.nature.com/nbt/journal/v3.../nbt.2938.html

    The PE primers they used are:
    PEprimerl AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
    PEprimer2 AAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT

    Should I tell sequencing center to use P5 and P7 primers for MiSeq for the PCR product? I can see P5 primer sequence in PEprimer1 but PEprimer2 doesn't have exact P7 sequence. It lacks first "C"? Could anyone help me?

    Thank you!

    Leave a comment:


  • pmiguel
    replied
    Originally posted by clueheart View Post
    Hi,

    i am uncertain what is the actual sequence of the read2 primer on Hiseq, PE amplicon sequencing.

    To my understanding it should be HP11.

    Is ATCTCGTATGCCGTCTTCTGCTTG the correct sequence for HP11 ?
    To the best of my knowledge Illumina never shares the sequences of their primers. Adapters, yes. Primers, no.

    --
    Phillip

    Leave a comment:


  • clueheart
    replied
    Hi,

    i am uncertain what is the actual sequence of the read2 primer on Hiseq, PE amplicon sequencing.

    To my understanding it should be HP11.

    Is ATCTCGTATGCCGTCTTCTGCTTG the correct sequence for HP11 ?

    Leave a comment:


  • Ingeneious
    replied
    Originally posted by ssully View Post
    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'     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?
    Code:
    5' AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATC-T-insert-A-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7]ATCTCGTATGCCGTCTTCTGCTTG 3'
    			      	                      |||||||||||||||||||||||||||||||||||
    3'     GTTCGTCTTCTGCCGTATGCTCTA[i7]CACTGACCTCAAGTCTGCACACGAGAAGGCTAG-A-insert-T-CTAGCCTTCTCGCAGCACATCCCTTTCTCACA[i5]CACATCTAGAGCCACCAGCGGCATAGTAA 5'

    Leave a comment:


  • ksil91
    replied
    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

    Leave a comment:


  • ssully
    replied
    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
    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:

    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.

    Leave a comment:


  • ssully
    replied
    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)
    Last edited by ssully; 11-17-2014, 07:22 PM.

    Leave a comment:

Latest Articles

Collapse

  • seqadmin
    An Introduction to the Technologies Transforming Precision Medicine
    by seqadmin


    In recent years, precision medicine has become a major focus for researchers and healthcare professionals. This approach offers personalized treatment and wellness plans by utilizing insights from each person's unique biology and lifestyle to deliver more effective care. Its advancement relies on innovative technologies that enable a deeper understanding of individual variability. In a joint documentary with our colleagues at Biocompare, we examined the foundational principles of precision...
    01-27-2025, 07:46 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, Yesterday, 09:30 AM
0 responses
16 views
0 likes
Last Post seqadmin  
Started by seqadmin, 02-05-2025, 10:34 AM
0 responses
28 views
0 likes
Last Post seqadmin  
Started by seqadmin, 02-03-2025, 09:07 AM
0 responses
27 views
0 likes
Last Post seqadmin  
Started by seqadmin, 01-31-2025, 08:31 AM
0 responses
35 views
0 likes
Last Post seqadmin  
Working...
X