SUPPLEMENT Table S1. Primers used in protocol

SUPPLEMENT Primer

Sequence

Uracil-containing A

GCGAN13UU

fragment primer Uracil-containing B

CCATN13UU

fragment primer A fragment uniqueF

CCATN13

B fragment uniqueR

GCGAN13

YF

GTTTTCCCAGTCACGAC

YR

CAGGAAACAGCTATGAC

Dial-Out_Tags_F

CGACAGTAACTACACGGCGAN13GTTTTCCCAGTCACGAC

Dial-Out_Tags_R

GTAGCAATTGGCAGGTCCATN13CAGGAAACAGCTATGAC

Dial-Out_Flow_Cell_F

AATGATACGGCGACCACCGAGATCTACACACGTAGGCCGA CAGTAACTACACGGCGA

Dial-Out_Flow_Cell_R

CAAGCAGAAGACGGCATACGAGATNNNNNNNNNGACCGT CGGCGTAGCAATTGGCAGGTCCAT

Dial-Out_Sequencing_F

ACGTAGGCCGACAGTAACTACACGGCGA

Dial-Out_Sequencing_R

GACCGTCGGCGTAGCAATTGGCAGGTCCAT

Dial-Out_Sequencing_I

ATGGACCTGCCAATTGCTACGCCGACGGTC

YF-pu1L

CTAAATGGCTGTGAGAGAGCTCAGGTTTTCCCAGTCACGAC

YF-pu1R

ACTTTATCAATCTCGCTCCAAACCCAGGAAACAGCTATGAC

Pu1L_Flow_Cell

AATGATACGGCGACCACCGAGATCTACACACGTAGGCCTA AATGGCTGTGAGAGAGCTCAG

Pu1R_Flow_Cell

CAAGCAGAAGACGGCATACGAGATNNNNNNNNNGACCGT CGGCACTTTATCAATCTCGCTCCAAACC

Pu1_Sequencing_F

ACGTAGGCCTAAATGGCTGTGAGAGAGCTCAG

Pu1_Sequencing_R

GACCGTCGGCACTTTATCAATCTCGCTCCAAACC

Pu1_Sequencing_I

GGTTTGGAGCGAGATTGATAAAGTGCCGACGGTC

Table S1. Primers used in protocol. The uracil-containing primers and Dial-Out tags both include insilico designed 13-mer barcodes, represented as N13 in this table. These were used for amplifying subpools from the array, as well as for tagging assembled constructs for Dial-Out PCR.

Polymerase

Tags in

%Yield (Targets with perfect

% of molecules with unique

moles

assemblies)

dial-out tag combination

Kapa HiFi

8.5E-14

91.3

81.5

Kapa HiFi

4.25E-13

91.3

85.7

Kapa HiFi

8.5E-13

84.6

89.3

Kapa HiFi

1.0E-12

90.3

-

Kapa 2G Robust

8.5E-14

64.4

-

Kapa 2G

8.5E-14

85.5

-

Multiplex

Table S2. Optimizing polymerase and tag concentration. We first tested the effect of several different tag concentrations on assembly yield with Kapa HiFi polymerase. For this dataset, the M13 sequences were present on the oligos, and tags were introduced during assembly. We found that we obtained the greatest yield with approximately a 10:1 molar ratio of tag:template, without a large loss in the percentage of unique tag pairs. We then tested this ratio with two different polymerases, Kapa 2G Robust and Kapa 2G Multiplex.

Multiplex Pairwise Assembly

Column-based synthesis

Raw oligo cost

$2,400

$99,776

Oligo Pool amplification with

$24

-

USER treatment + End Repair

$36

-

Assembly PCR

$12

-

Sequence Verification

$150

-

Total Cost

$2,622

$99,776

Dial-Out Tag Library- one-time

$1,800

-

$17,118

-

Kapa HiFi Uracil+

cost Retrieval Primer Library- one-time cost Dial-Out: Total one-time cost

$18,918

Dial-Out Tagging and Sequencing

$150

Dial-Out Retrieval

$3,118

Total Cost with Dial-Out

$5,890

-

Retrieval

Table S3. Cost breakdown for 3,118 200-mers. Raw oligo cost for multiplex pairwise assembly is based on duplicating all oligos and filling one 12,472-array from CustomArray with 160mers. Column-based oligo cost is based on IDT price of 384-well sub-nanomole plates. Note that IDT cannot synthesis oligos longer than 200bp by this method, whereas we demonstrate the synthesis of 252-mers. The rest of the steps for multiplex pairwise assembly are based on separating targets into six pools. Sequencing costs are based on a MiSeq v2 300 cycle spike-in (2 million reads). For Dial-Out Tagging, there is a one-time cost of the tag and PCR retrieval libraries. The total cost with Dial-Out Retrieval does not include the one-time cost.

Figure S1. Comparison of one vs. two unique primers per oligo pool. We amplified pools of oligos off the array using either one unique primer (Uracil-containing A/B fragment primer) and one common primer (YF/YR), or two unique primers (Uracil-containing A/B fragment primer and A/B fragment uniqueF/R) (Supplemental Table S1). We then assembled each pool and sequenced to 115,000 reads. A) Percentage of perfect, mismatch only, small indels (