Sequencing Database and Barcodes

Required!

Use the linkeddatabase to generate a sample name with a unique id and to track associated meta data.

Data base instructions

To be updated:

  • Arrange access through the lab manager or Ty.
  • Do not delete other peoples entries!
  • Highlighted columns are required and used for generating sample names.
  • Use generated sample names in submitted sample sheets.
  • Track relevant meta data in additional columns.

Lab Sequencing Barcodes

Important
  • Not all barcode (index) sequences between manufacturers are compatible with each other. A partial list of our colliding barcodes (indexes) can be found on the lab GitHub.
  • It is important to plan ahead, consider Hamming distances, GC balance, and communicate with colleagues to facilitate multiplexing.
  • Examples for determining Hamming distances can be found on the lab GitHub.
  • Barcode (index) lists are also available on the lab GitHub.

NEXTFLEX UDI Barcodes

Important
  • Never thaw the annealed adapters above room temperature or they can denature.
  • Mix carefully and centrifuge prior to opening.
  • Handle carefully to avoid downstream adapter cross-contamination.

We have three sets of NEXTFLEX full-length unique dual index (UDI) barcoding adapters from Perkin Elmer. These adapters are for use in ligation chemistry (lab standard dUTP RNA-seq and ChIP-seq protocols). They contain a unique 8 nucleotide index in the P5 (i5) and P7 (i7) sequence.

These UDI barcodes are purchased in sets of 96 adapters. We purchased set D (barcodes 289:384) and were gifted small aliquots of set A (1:96) and set B (97:192) from the labs’ of Chris Glass and Sven Heinz at UC San Diego.

Each pair was designed with a Hamming distance of at least 3 across the entire series of 384 UDI barcodes, meaning any two or more chosen NEXTFLEX UDI adapters in the entire set are internally compatible with themselves. They are not all appropriately GC balanced though. Each consecutive pair of barcodes found in columns 1 and 2 are fully color balanced and are suitable to be used in a pool of two samples. When designing low-plexity index pools, always include two libraries barcoded with a set of two unique and fully color balanced barcodes to avoid laser color complexity issues during de-multiplexing.

Adapter stocks are delivered at 25 µM and diluted to 0.625 µM in 1X T4 DNA ligase buffer (10X T4 ligase buffer, NEB #B0202S; dilute 1:10 with ultra-pure water to obtain 1X buffer). Diluted stocks are further diluted to 0.25 µM as routine use aliquots. Typically, 0.5 to 1 µl of the working aliquot is adequate for adapter ligation. Barcoded libraries are amplified using NEXTFLEX common primer sequences.

NEXTFLEX PCR Primer 1: AATGATACGGCGACCACCGAGATCTACAC 
NEXTFLEX PCR Primer 2: CAAGCAGAAGACGGCATACGAGAT

NEXTFLEX i5 Adapter: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCT
NEXTFLEX i7 Adapter: GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXXXATCTCGTATGCCGTCTTCTGCTTG
Note

XXXXXXXX denotes the i5 or i7 index region of the adapter.

‘Nextera’ Tn5 Barcoding Primers

Buenrostro et. al. Nature, 2015. PMID: 26083756.

Nextera Tn5 transposase barcoding primers for (ATAC-seq and other suitable transposase chemistry libraries) were sourced from the labs’ of Howard Chang and William Greenleaf (Supplementary Table 1 of the linked manuscript). Each primer contains a unique 8 nucleotide index. Combinatorial or unique i7 and i7 primers are selected for each sample and are used as primers for PCR amplification of ATAC-seq libraries.

Primers were ordered from IDT as standard oligonucleotides and suspended at 10 µM in low-EDTA TE buffer. Reconstituted primers stocks are stored at -20C. Working aliquots should be used routinely in place of the stocks.

Nextera Tn5 i5 Primer Sequence: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXTCGTCGGCAGCGTCAGATGTGTAT
Nextera Tn5 i7 Primer Sequence: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTCTCGTGGGCTCGGAGATGTG
Note

XXXXXXXX denotes the i5 or i7 index region of the primer.

Additional information

For barcoded adapters added via ligation (e.g. NEXTFLEX UDI, standard TruSeq style Y-adapters):
  • Index 1 (i7) is always read in the direction (5’ to 3′) of the sequence in the oligo.
  • index 2 (i5) is read in the direction of the oligo for Miseq and NovaSeq v1 chemistry.
  • Index 2 (i5) is read as reverse complement of barcoded PCR oligos for NextSeq, iSeq, and NovaSeq using the version 1.5 (reverse complement) chemistry. The CCHMC DNA Sequencing core is using version 1.5 chemistry for the NovaSeq.
For barcodes added via a PCR step (e.g. Tn5 Transposase Barcoding PCR Primers, Nextera; or through primers and stubby adapters):
  • Index 1 (i7) is always read as the reverse complement of the sequence in the TruSeq or Nextera style PCR oligos. The PCR primer sequence is the reverse complement of the product it generates. Don’t trust Ty to explain this to you.
  • Index 2 (i5) is read in the direction of the TruSeq or Nextera PCR oligos for Miseq and NovaSeq runs (v1 chemistry).
  • Index 2 (i5) is read as the reverse complement of barcoded PCR oligos for NextSeq, iSeq, and NovaSeq (v1.5 chemistry) runs.