A Sample List of 96 Functional Barcodes
IMPORTANT: barcodes and Illumina HiSeq
We find that the Illumina Hiseq is more temperamental about barcodes than the GAII, perhaps because of the lower intrinsic resolution provided by scanning vs. photographing. The first 5 cycles of Illumina are used to discriminate the clusters: two very close clusters will be treated as one if they display the same sequence. However, if they diverge after the fifth cycle the sequencing will fail or be of low quality. Additionally, the second cycle is used to measure the overlap between fluorescent channels. This measurement expects roughly equal number of C and A clusters. Bias at this level will compromise the run. You may be able to rescue these runs by rerunning the analysis after cutting off the first 5 cycles. As a result of these requirements, increasing cluster density is not well tolerated if few barcodes are used (such as 4). To be on the safe side, we use at least 16 barcodes per Hiseq lane.
For RNAseq and pools that combine less than 24 samples, we are now using the Illumina-compatible index system. For example, we have had good results with these adapters. However, we do not endorse or guarantee this product and if you try them, it is at your own “risk”. Just in case, you may want to dilute them 1/10 beyond the recommended concentration and see if they work for you.
Processing barcoded reads
We provide a set of scripts for procession barcoded reads
The Barcodes
These barcodes and associated oligonucleotides were generated using our barcode generator. For more background information on barcoding, see our instructional videos on Illumina Multiplexing. We order our adapters from Life Tech using desalted purity. Make master stocks in TE and keep them at -80 C.
This sample list is also available:
NOMENCLATURE: By convention, the barcode is named for the sequence found on the adA adaptor. However the barcode found in the actual Illumina sequence will be the reverse complement of this.
Index of Barcodes (Length of 5, distance between barcodes of 2 bases):
| aaaca | aaatc | aacat | aagag | aagcc | aagtt | aatgc | acact | acccc | accgg | acgca | acgtg |
| actaa | actcg | actgt | agcga | agctt | aggac | agtca | agtgg | atacg | atagc | atcac | atgta |
| attcc | attga | caaaa | caatt | caccc | catct | cattc | cccca | ccctt | ccgag | cgaat | cgagg |
| cgatc | cgcac | cgcgt | cggtt | cgttg | ctaag | ctcga | ctctc | ctgcc | ctggt | cttgg | gaagc |
| gactt | gagca | gatac | gatga | gcaaa | gccat | gccta | gcgtt | gctcc | gctgg | ggaac | ggatg |
| ggcag | gggaa | ggtgt | gtacc | gtagt | gtata | gtcaa | gtcct | gtctg | gtgag | gtgga | gttat |
| gttca | gtttc | taaga | tactg | tagct | tatag | tatcc | tcatt | tccag | tccct | tcgac | tcgcg |
| tcgga | tgaag | tgacc | tgata | tggat | tggtc | tgtcg | tgtgc | tgttt | ttact | ttagg | ttccc |
List of Oligonucleotides
Important Note: adA needs to be 5′ phosphorylated
>adA_aaaca
P-aaacaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aaaca
ACACTCTTTCCCTACACGACGCTCTTCCGATCTtgtttT
>adA_aaatc
P-aaatcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aaatc
ACACTCTTTCCCTACACGACGCTCTTCCGATCTgatttT
>adA_aacat
P-aacatAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aacat
ACACTCTTTCCCTACACGACGCTCTTCCGATCTatgttT
>adA_aagag
P-aagagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aagag
ACACTCTTTCCCTACACGACGCTCTTCCGATCTctcttT
>adA_aagcc
P-aagccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aagcc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggcttT
>adA_aagtt
P-aagttAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aagtt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaacttT
>adA_aatgc
P-aatgcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aatgc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgcattT
>adA_acact
P-acactAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_acact ACACTCTTTCCCTACACGACGCTCTTCCGATCTagtgtT
>adA_acccc
P-accccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_acccc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggggtT
>adA_accgg
P-accggAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_accgg ACACTCTTTCCCTACACGACGCTCTTCCGATCTccggtT
>adA_acgca
P-acgcaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_acgca ACACTCTTTCCCTACACGACGCTCTTCCGATCTtgcgtT
>adA_acgtg
P-acgtgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_acgtg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcacgtT
>adA_actaa
P-actaaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_actaa ACACTCTTTCCCTACACGACGCTCTTCCGATCTttagtT
>adA_actcg
P-actcgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_actcg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcgagtT
>adA_actgt
P-actgtAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_actgt ACACTCTTTCCCTACACGACGCTCTTCCGATCTacagtT
>adA_agcga
P-agcgaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_agcga ACACTCTTTCCCTACACGACGCTCTTCCGATCTtcgctT
>adA_agctt
P-agcttAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_agctt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaagctT
>adA_aggac
P-aggacAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_aggac ACACTCTTTCCCTACACGACGCTCTTCCGATCTgtcctT
>adA_agtca
P-agtcaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_agtca ACACTCTTTCCCTACACGACGCTCTTCCGATCTtgactT
>adA_agtgg
P-agtggAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_agtgg ACACTCTTTCCCTACACGACGCTCTTCCGATCTccactT
>adA_atacg
P-atacgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_atacg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcgtatT
>adA_atagc
P-atagcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_atagc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgctatT
>adA_atcac
P-atcacAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_atcac ACACTCTTTCCCTACACGACGCTCTTCCGATCTgtgatT
>adA_atgta
P-atgtaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_atgta ACACTCTTTCCCTACACGACGCTCTTCCGATCTtacatT
>adA_attcc
P-attccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_attcc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggaatT
>adA_attga
P-attgaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_attga ACACTCTTTCCCTACACGACGCTCTTCCGATCTtcaatT
>adA_caaaa
P-caaaaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_caaaa ACACTCTTTCCCTACACGACGCTCTTCCGATCTttttgT
>adA_caatt
P-caattAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_caatt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaattgT
>adA_caccc
P-cacccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_caccc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgggtgT
>adA_catct
P-catctAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_catct ACACTCTTTCCCTACACGACGCTCTTCCGATCTagatgT
>adA_cattc
P-cattcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cattc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgaatgT
>adA_cccca
P-ccccaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cccca ACACTCTTTCCCTACACGACGCTCTTCCGATCTtggggT
>adA_ccctt
P-cccttAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ccctt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaagggT
>adA_ccgag
P-ccgagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ccgag ACACTCTTTCCCTACACGACGCTCTTCCGATCTctcggT
>adA_cgaat
P-cgaatAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cgaat ACACTCTTTCCCTACACGACGCTCTTCCGATCTattcgT
>adA_cgagg
P-cgaggAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cgagg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcctcgT
>adA_cgatc
P-cgatcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cgatc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgatcgT
>adA_cgcac
P-cgcacAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cgcac ACACTCTTTCCCTACACGACGCTCTTCCGATCTgtgcgT
>adA_cgcgt
P-cgcgtAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cgcgt ACACTCTTTCCCTACACGACGCTCTTCCGATCTacgcgT
>adA_cggtt
P-cggttAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cggtt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaaccgT
>adA_cgttg
P-cgttgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cgttg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcaacgT
>adA_ctaag
P-ctaagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ctaag ACACTCTTTCCCTACACGACGCTCTTCCGATCTcttagT
>adA_ctcga
P-ctcgaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ctcga ACACTCTTTCCCTACACGACGCTCTTCCGATCTtcgagT
>adA_ctctc
P-ctctcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ctctc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgagagT
>adA_ctgcc
P-ctgccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ctgcc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggcagT
>adA_ctggt
P-ctggtAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ctggt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaccagT
>adA_cttgg
P-cttggAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_cttgg ACACTCTTTCCCTACACGACGCTCTTCCGATCTccaagT
>adA_gaagc
P-gaagcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gaagc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgcttcT
>adA_gactt
P-gacttAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gactt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaagtcT
>adA_gagca
P-gagcaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gagca ACACTCTTTCCCTACACGACGCTCTTCCGATCTtgctcT
>adA_gatac
P-gatacAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gatac ACACTCTTTCCCTACACGACGCTCTTCCGATCTgtatcT
>adA_gatga
P-gatgaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gatga ACACTCTTTCCCTACACGACGCTCTTCCGATCTtcatcT
>adA_gcaaa
P-gcaaaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gcaaa ACACTCTTTCCCTACACGACGCTCTTCCGATCTtttgcT
>adA_gccat
P-gccatAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gccat ACACTCTTTCCCTACACGACGCTCTTCCGATCTatggcT
>adA_gccta
P-gcctaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gccta ACACTCTTTCCCTACACGACGCTCTTCCGATCTtaggcT
>adA_gcgtt
P-gcgttAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gcgtt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaacgcT
>adA_gctcc
P-gctccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gctcc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggagcT
>adA_gctgg
P-gctggAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gctgg ACACTCTTTCCCTACACGACGCTCTTCCGATCTccagcT
>adA_ggaac
P-ggaacAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ggaac ACACTCTTTCCCTACACGACGCTCTTCCGATCTgttccT
>adA_ggatg
P-ggatgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ggatg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcatccT
>adA_ggcag
P-ggcagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ggcag ACACTCTTTCCCTACACGACGCTCTTCCGATCTctgccT
>adA_gggaa
P-gggaaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gggaa ACACTCTTTCCCTACACGACGCTCTTCCGATCTttcccT
>adA_ggtgt
P-ggtgtAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ggtgt ACACTCTTTCCCTACACGACGCTCTTCCGATCTacaccT
>adA_gtacc
P-gtaccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtacc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggtacT
>adA_gtagt
P-gtagtAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtagt ACACTCTTTCCCTACACGACGCTCTTCCGATCTactacT
>adA_gtata
P-gtataAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtata ACACTCTTTCCCTACACGACGCTCTTCCGATCTtatacT
>adA_gtcaa
P-gtcaaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtcaa ACACTCTTTCCCTACACGACGCTCTTCCGATCTttgacT
>adA_gtcct
P-gtcctAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtcct ACACTCTTTCCCTACACGACGCTCTTCCGATCTaggacT
>adA_gtctg
P-gtctgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtctg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcagacT
>adA_gtgag
P-gtgagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtgag ACACTCTTTCCCTACACGACGCTCTTCCGATCTctcacT
>adA_gtgga
P-gtggaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtgga ACACTCTTTCCCTACACGACGCTCTTCCGATCTtccacT
>adA_gttat
P-gttatAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gttat ACACTCTTTCCCTACACGACGCTCTTCCGATCTataacT
>adA_gttca
P-gttcaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gttca ACACTCTTTCCCTACACGACGCTCTTCCGATCTtgaacT
>adA_gtttc
P-gtttcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_gtttc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgaaacT
>adA_taaga
P-taagaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_taaga ACACTCTTTCCCTACACGACGCTCTTCCGATCTtcttaT
>adA_tactg
P-tactgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tactg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcagtaT
>adA_tagct
P-tagctAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tagct ACACTCTTTCCCTACACGACGCTCTTCCGATCTagctaT
>adA_tatag
P-tatagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tatag ACACTCTTTCCCTACACGACGCTCTTCCGATCTctataT
>adA_tatcc
P-tatccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tatcc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggataT
>adA_tcatt
P-tcattAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tcatt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaatgaT
>adA_tccag
P-tccagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tccag ACACTCTTTCCCTACACGACGCTCTTCCGATCTctggaT
>adA_tccct
P-tccctAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tccct ACACTCTTTCCCTACACGACGCTCTTCCGATCTagggaT
>adA_tcgac
P-tcgacAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tcgac ACACTCTTTCCCTACACGACGCTCTTCCGATCTgtcgaT
>adA_tcgcg
P-tcgcgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tcgcg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcgcgaT
>adA_tcgga
P-tcggaAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tcgga ACACTCTTTCCCTACACGACGCTCTTCCGATCTtccgaT
>adA_tgaag
P-tgaagAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tgaag ACACTCTTTCCCTACACGACGCTCTTCCGATCTcttcaT
>adA_tgacc
P-tgaccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tgacc ACACTCTTTCCCTACACGACGCTCTTCCGATCTggtcaT
>adA_tgata
P-tgataAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tgata ACACTCTTTCCCTACACGACGCTCTTCCGATCTtatcaT
>adA_tggat
P-tggatAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tggat ACACTCTTTCCCTACACGACGCTCTTCCGATCTatccaT
>adA_tggtc
P-tggtcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tggtc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgaccaT
>adA_tgtcg
P-tgtcgAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tgtcg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcgacaT
>adA_tgtgc
P-tgtgcAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tgtgc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgcacaT
>adA_tgttt
P-tgtttAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_tgttt ACACTCTTTCCCTACACGACGCTCTTCCGATCTaaacaT
>adA_ttact
P-ttactAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ttact ACACTCTTTCCCTACACGACGCTCTTCCGATCTagtaaT
>adA_ttagg
P-ttaggAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ttagg ACACTCTTTCCCTACACGACGCTCTTCCGATCTcctaaT
>adA_ttccc
P-ttcccAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
>adB_ttccc ACACTCTTTCCCTACACGACGCTCTTCCGATCTgggaaT
Comai Lab 