| IS1_adapter.P5 |
A*C*A*C*TCTTTCCCTACACGACGCTCTTCCG*A*T*C*T |
| IS2_adapter.P7 |
G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCG*A*T*C*T |
| IS3_adapter.P5+P7 |
A*G*A*T*CGGAA*G*A*G*C |
| IS4_indPCR.P5 |
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT |
| IS5_reamp.P5 |
AATGATACGGCGACCACCGA |
| IS6_reamp.P7 |
CAAGCAGAAGACGGCATACGA |
| IS7_short_amp.P5 |
ACACTCTTTCCCTACACGAC |
| IS8_short_amp.P7 |
GTGACTGGAGTTCAGACGTGT |
| BO1.P5.F |
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-Pho |
| BO2.P5.R |
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT-Pho |
| BO3.P7.part1.F |
AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-Pho |
| BO4.P7.part1.R |
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-Pho |
| BO5.P7.part2.F |
ATCTCGTATGCCGTCTTCTGCTTG-Pho |
| BO6.P7.part2.R |
CAAGCAGAAGACGGCATACGAGAT-Pho |
|
| a5′-3′; * indicates a PTO bond; Pho indicates a 3′-phosphate.
|
| See Supplemental Material (Indexing_Oligo_Sequences.doc) for indexing oligo sequences. |
| All oligos (HPLC purified, 0.2 μmol synthesis scale) should be dissolved in TE or H2O. Oligos 1-3 should be dissolved to 500 μM, oligos BO1-BO6 to 200 μM, and all other oligos to 10 μM. The indexing oligos
should be transferred to a 96-well plate to allow for multichannel pipetting. HPLC purification can potentially introduce
cross-contamination among indexing oligos. It is therefore important to (1) instruct the company to properly wash the HPLC
column before loading a new oligo and (2) synthesize the oligos in a different order than listed here. This makes sure that
cross-contamination induced during synthesis can be detected after sequencing by the appearance of index sequences that were
not used in the experiment. When designing index sequences, the following criteria were taken into account: (1) Index sequences
differ by at least three substitutions. This reduces the chance of converting one index into another by sequencing and amplification
errors. (2) Indexes cannot be converted into one another by deleting the first base, which is the only insertion/deletion
error common with Illumina sequencing. (3) Index sequences do not contain three or more identical bases in a row to ensure
that they can be differentiated from artifact sequences. (4) Stretches of bases illuminated with the same laser (ACA, CAC,
GTG, and TGT) are avoided. Software for designing alternative index sequences, for example, with a length of 6 or 8 nt, and
for selecting appropriate subsets for pooling is provided at http://bioinf.eva.mpg.de.
|
