Illumina GA/Oligo info: Difference between revisions

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* May 2013, more NEBNext Index primers (Index 9-32) in 96-well plate were ordered. The new primers do not have phosphorothioate bond at -s-T-3´ end as the ones from NEB kit.
* May 2013, more NEBNext Index primers (Index 9-32) in 96-well plate were ordered. The new primers do not have phosphorothioate bond at -s-T-3´ end as the ones from NEB kit.
* Updated [[Media:ZhangLab_NEBNext_MultiplexingPrimers_BarcodeInfo_06142013.xlsx| ZhangLab_NEBNext_MultiplexingPrimers_BarcodeInfo_06142013]]
* Updated [[Media:ZhangLab_NEBNext_MultiplexingPrimers_BarcodeInfo_06142013.xlsx| ZhangLab_NEBNext_MultiplexingPrimers_BarcodeInfo_06142013]]
==HiTSFLIP Sequencing Primer==
*HiTSFLIP SEQ 1 5'GATCACGACGCTCTTCCGATCTCTACACTCTTTCCCTA
*Multiplexing Index Read Sequencing Primer 5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

Revision as of 19:59, 3 September 2013

Name in original paper Nick-name Sequence (5\'->3\') Sequence Modification Reference
A_adapter_t Long Y-adaptor (top) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond [1]
A_adapter_b Long Y-adaptor (bottom) GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG None [1]
PE_t_adapter Regular Y-adaptor (top) ACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond [2]
PE_b_adapter Regular Y-adaptor (bottom) /5Phos/GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-phosphorylation [2]
PCR_F Sol. Amp. (Forward) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond [2]
PCR_R Sol. Amp. (Reverse) CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T 3'-Phosphorothioate bond [2]
Syb_FP5 Sol. Quant. (Forward) ATGATACGGCGACCACCGAG None [2]
Syb_RP7 Sol. Quant. (Reverse) CAAGCAGAAGACGGCATACGAG None [2]
PE_t_N2 Y-adaptor for MmeI ligation (top) ACACTCTTTCCCTACACGACGCTCTTCCGATCTN*N 3'-Phosphorothioate bond
PE_N2 Y-adaptor for MmeI ligation (top) ACACTCTTTCCCTACACGACGCTCTTCCGATC*NN 3'-Phosphorothioate bond
PE_b_A Y-adaptor for MmeI ligation (bottom) /5Phos/AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-phosphorylation
PE_t_Met bis-seq(Forward) A/iMe-dC/A/iMe-dC/T/iMe-dC/TTT/iMe-dC//iMe-dC//iMe-dC/TA/iMe-dC/ACGACG/iMe-dC/T/iMe-dC/TT/iMe-dC/CGAT/iMe-dC/*T 3-phosphorothiate, 5'-Methyl
PE_b_Met bis-seq (Reverse) /5Phos/GATCGGAAGAGCGGTT/iMe-dC/AG/iMe-dC/AGGAATG/iMe-dC/CGAG 5'-phosphorylated, 5'-Methyl
RC_PE_t_adapter RC_Regular Y-adaptor with Biotin (top) /5Biosg/TTTTTTTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 5'-Biotin
RC_PE_b_adapter RC_Regular Y-adaptor with Biotin (bottom) /5Biosg/TTTTTTTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC 5'-Biotin
Small RNA Sequencing Primer Solexa-TSS-Seq CGACAGGTTCAGAGTTCTACAGTCCGACGATC

Multiplexing Reverse PCR primers

Library Free Multiplexing Primers

Name Nick-name Sequence (5\'->3\') Sequence Modification Reference
Left Probe Linker, No barcode (forward) AmpF6.3Sol AATGATACGGCGACCACCGACACTCTCAGATGTTATCGAGGTCCGAC None None
Right Probe Linker, Barcode 1: CGTGAT (reverse) AmpR6.3Ind1 CAAGCAGAAGACGGCATACGAGATCGTGATGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 2: ACATCG (reverse) AmpR6.3Ind2 CAAGCAGAAGACGGCATACGAGATACATCGGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 3: GCCTAA (reverse) AmpR6.3Ind3 CAAGCAGAAGACGGCATACGAGATGCCTAAGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 4: TGGTCA (reverse) AmpR6.3Ind4 CAAGCAGAAGACGGCATACGAGATTGGTCAGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 5: CACTGT (reverse) AmpR6.3Ind5 CAAGCAGAAGACGGCATACGAGATCACTGTGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 6: ATTGGC (reverse) AmpR6.3Ind6 CAAGCAGAAGACGGCATACGAGATATTGGCGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 7: GATCTG (reverse) AmpR6.3Ind7 CAAGCAGAAGACGGCATACGAGATGATCTGGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 8: TCAAGT (reverse) AmpR6.3Ind8 CAAGCAGAAGACGGCATACGAGATTCAAGTGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 9: CTGATC (reverse) AmpR6.3Ind9 CAAGCAGAAGACGGCATACGAGATCTGATCGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 10: AAGCTA (reverse) AmpR6.3Ind10 CAAGCAGAAGACGGCATACGAGATAAGCTAGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 11: GTAGCC (reverse) AmpR6.3Ind11 CAAGCAGAAGACGGCATACGAGATGTAGCCGCTAGGAACGATGAGCCTCCAAC None None
Right Probe Linker, Barcode 12: TACAAG (reverse) AmpR6.3Ind12 CAAGCAGAAGACGGCATACGAGATTACAAGGCTAGGAACGATGAGCCTCCAAC None None
Forward sequencing primer SolSeqV6.3.2 ACCGACACTCTCAGATGTTATCGAGGTCCGAC None None
Reverse sequencing primer SolSeqV6.3.2r GCTAGGAACGATGAGCCTCCAAC None None
Index read primer AmpR6.3IndSeq GTTGGAGGCTCATCGTTCCTAGC None None
Forward extension primer AmpF6.3SolExt AATGATACGGCGACCACCGAGATCTACACCACTCTCAGATGTTATCGA None None
Forward PCR primer AmpF6.4Sol AATGATACGGCGACCACCGAGATCTACACCACTCTCAGATGTTATCGAGGTCCGAC None None
Forward sequencing primer SolSeqV6.3.3 TACACCACTCTCAGATGTTATCGAGGTCCGAC None None

Multiplex primers for the N2-adaptor

Sequence ID Sequence
PCR_R.N2Ind1 CAAGCAGAAGACGGCATACGAGATCGTGATCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind2 CAAGCAGAAGACGGCATACGAGATACATCGCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind3 CAAGCAGAAGACGGCATACGAGATGCCTAACTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind4 CAAGCAGAAGACGGCATACGAGATTGGTCACTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind5 CAAGCAGAAGACGGCATACGAGATCACTGTCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind6 CAAGCAGAAGACGGCATACGAGATATTGGCCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind7 CAAGCAGAAGACGGCATACGAGATGATCTGCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind8 CAAGCAGAAGACGGCATACGAGATTCAAGTCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind9 CAAGCAGAAGACGGCATACGAGATCTGATCCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind10 CAAGCAGAAGACGGCATACGAGATAAGCTACTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind11 CAAGCAGAAGACGGCATACGAGATGTAGCCCTCGGCATTCCTGCTGAACCGCTCTT
PCR_R.N2Ind12 CAAGCAGAAGACGGCATACGAGATTACAAGCTCGGCATTCCTGCTGAACCGCTCTT
PCR_Fs AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC
N2IndSeq AAGAGCGGTTCAGCAGGAATGCCGAG
N2RevSeq(WRONG design) CTCGGCATTCCTGCTGAACCGCTCTT
N2RevSeq2 CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
N2FwdSeq2H (for overloaded flowcells only) CACTCTTTCCCTACACGACGCTCTTCCGATCTH
N2RevSeq2H (for overloaded flowcells only) TCGGCATTCCTGCTGAACCGCTCTTCCGATCTH

Nextera Read Primer

Sequence ID Sequence
Nextera Read 1 Primer 5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3'
Nextera Read 2 Primer 5'-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG-3'
Nextera Index Read Primer 5'-CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG-3'


Nextera Primer

ID Sequence Length
Nextera Primer 1* AATGATACGGCGACCACCGA 20nt
Nextera Primer 2* CAAGCAGAAGACGGCATACGA 21nt
Nextera Adaptor 1* AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG 48nt
Nextera Adaptor 2 (minus bar code)* CAAGCAGAAGACGGCATACGAGATCGGTCTGCCTTGCCAGCCCGCTCAG 49nt
Orange primer CCTTGCCAGCCCGCTCAG 18nt
Blue primer CCTCCCTCGCGCCATCAG 18nt
Short Orange primer CCTTGCCAGCC 11nt
Short Blue primer CCTCCCTCGCG 11nt

Primers for BSPP probes synthesized by Agilent

Sequence ID Sequences Length
pAP1V61U 5’-G*G*G*TCATATCGGTCACTGTU-3’ 18
AP1V6 5\'- GTCATATCGGTCACTGTT -3\' 18
AP2V6 5’-/5Phos/CACGGGTAGTGTGTATCCTG-3’ 20
  • * Indicates a phosphorothioate bond

Primers for LC Sciences probe experiment

Sequence ID Sequnces Length
eMIP_CA1_F TGCCTAGGACCGGATCAACT 20
eMIP_CA1_R GAGCTTCGGTTCACGCAATG 20
CP-2-FA GCACGATCCGACGGTAGTGT 20
CP-2-RA CCGTAATCGGGAAGCTGAAG 20
CP-2-FA.IndSol CAAGCAGAAGACGGCATACGAGATNNNNNNCGGTCTGCCATCCGACGGTAGTGT 54
CP-2-RA.Sol AATGATACGGCGACCACCGAGATCTACACGCCTATCGGGAAGCTGAAG 48
Read 1: CP-2-SeqRead1.x TACACGCCTATCGGGAAGCTGAAG 24
Index Read: CP-2-IndSeq.x ACACTACCGTCGGATGGCAGACCG 24
Switch.CP-2-FA.Sol AATGATACGGCGACCACCGAGATCTACACGCCTATCCGACGGTAGTGT 48
Switch.CP-2-RA.IndSol CAAGCAGAAGACGGCATACGAGATNNNNNNCGGTCTGCCATCGGGAAGCTGAAG 54
Read 1 CP-2-SeqRead1.y TACACGCCTATCCGACGGTAGTGT 24
Index Read:CP-2-IndSeq.y CTTCAGCTTCCCGATGGCAGACCG 24


Primers for LC Sciences library-free protocol

Sequence ID Sequences Length
CA-2-FA.Indx7Sol 5’-CAAGCAGAAGACGGCATACGAGATGATCTGCGGTCTGCCATCCGACGGTAGTGT-3’ 54
CA-2-FA.Indx45Sol 5'-CAAGCAGAAGACGGCATACGAGATCGTAGTCGGTCTGCCATCCGACGGTAGTGT-3' 54
CA-2-FA.Indx76Sol 5'-CAAGCAGAAGACGGCATACGAGATAATAGGCGGTCTGCCATCCGACGGTAGTGT-3' 54
CA-2-FA.Indx77Sol 5'-CAAGCAGAAGACGGCATACGAGATACCCGTCGGTCTGCCATCCGACGGTAGTGT-3' 54
CA-2-FA.Indx78Sol 5'-CAAGCAGAAGACGGCATACGAGATAACACGCGGTCTGCCATCCGACGGTAGTGT-3' 54
CA-2-RA.Sol 5’-AATGATACGGCGACCACCGAGATCTACACGCCTATCGGGAAGCTGAAG-3’ 48
CP-2-SeqRead1.x (Read1) 5’-TACACGCCTATCGGGAAGCTGAAG-3’ 24
CP-2-SeqRead2 5'-CGGTCTGCCATCCGACGGTAGTGT-3' 24
CP-2-IndSeq.x (IndexRead) 5’-ACACTACCGTCGGATGGCAGACCG-3’ 24


Primers for totoRNAseq protocol

totoRNAseq Read 1: [1]

CACCGAGATCTACACTCTTTCCCTACACGACGTT
34nt 	Tm=64(IDT)	Tm=71.72(Kun's)
correspond to TSO.idXX primers used for 5'end

totoRNAseq Read 1 v2:

CCACCGAGATCTACACTCTTTCCCTACACGACG
33nt 	Tm=64.5(IDT)	Tm=72.04(Kun's)
correspond to TSO.r02/TSO.r04 primers used for 5'end

RL.T7.IndSeq:

AAAAAAAAAAAAAAAAAAAAGCGGGCTGGCAAGG
34nt 	Tm=60.7(IDT)	Tm=68.5(Kun's)
correspond to RL.T7.id01-24 primers used for RT step (T20 based primers)

Primers for NEBNext Multiplex Oligos (updated May 08, 2013)

NEBNext adaptor for Illumina: 5´-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC/ideoxyu/ACACTCTTTCCCT ACACGACGCTCTTCCGATC*T-3´ (loop adaptor, commercially purchased for ultra-low input ligation)
Sequence ID Sequences
NEBNext Universal PCR Primer (PCR_F) 5´-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´
NEBNext Index 1 5´-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 2 5´-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 3 5´-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 4 5´-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 5 5´-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 6 5´-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3'
NEBNext Index 7 5´-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 8 5´-CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 9 5´-CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 10 5´-CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 11 5´-CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
NEBNext Index 12 5´-CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
HP1 -> updated to HP6 (read1)
NEBNextRevSeq2 (read2) 5' -GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT- 3'
NEBNextIndSeq (index read) 5' -GAAGAGCACACGTCTGAACTCCAGTCAC- 3'

HiTSFLIP Sequencing Primer

  • HiTSFLIP SEQ 1 5'GATCACGACGCTCTTCCGATCTCTACACTCTTTCCCTA
  • Multiplexing Index Read Sequencing Primer 5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC