Systematic benchmarking of single-cell ATAC-sequencing protocols
). This dimension-reduced representation exhibited a radial axis of ‘quality’ centered around the bottom lefthand corner and showed that technical replicate experiments produced highly similar datasets.
We found sequencing efficiency to be generally low for scATAC-seq experiments. Two strategies could mitigate this issue: optimized sample preparation and nuclei extraction protocols to minimize the amount of ambient chromatin in samples, potentially applying FACS for sample cleanup, and sequencing below library saturation to limit the number of duplicate reads.
Samples v1 V1 and v1 V2 were sequenced on a NovaSeq 6000 using a NovaSeq SP kit , and sequencing was performed using the following read protocol: 50 cycles , 8 cycles , 16 cycles and 49 cycles .PBMCs were thawed, and nuclei were isolated as described above for samples v1.1 C1–v1.1 C3 and v1.1 T1. For samples v1.1 St1 and v1.1 St2, a different thawing/isolation protocol was used.
Samples v2 v1 and v2 v2 were sequenced on an Illumina NextSeq 2000 under the following sequencing conditions: 50 bp , 8 bp , 16 bp and 50 bp . Samples v2 C1, v2 C2, v2 T1 and v2 T2 were sequenced on an Illumina NovaSeq 6000 under the following sequencing conditions: 50 bp , 8 bp , 16 bp and 49 bp .PBMCs were thawed as described above. The isolation of nuclei was slightly different, including the use of RNase inhibitors to ensure RNA quality.
Samples mt C1 and mt C2 were sequenced on an Illumina NovaSeq 6000 under the following sequencing conditions: 50 bp , 8 bp , 16 bp and 49 bp . Samples mt M1 and mt M2 were sequenced on an Illumina NovaSeq 6000 under the following sequencing conditions: 150 bp , 8 bp , 16 bp and 150 bp . Samples mt* Br1 and mt* Br2 were sequenced on an Illumina Nextseq 550 with paired-end reads , 8 cycles for index 1 and 16 cycles for index 2.
For samples ddS H1, ddS H2, ddS Bi3, ddS Bi4, ddS U1 and ddS U2, libraries were sequenced on a NextSeq 550 using a NextSeq High-Output kit and the following read protocol: 118 cycles , 8 cycles and 40 cycles . For ddS Bi1 and ddS Bi2, samples were sequenced on a NovaSeq according to the same protocol. A custom sequencing primer was required for read 1 .PBMCs were thawed, and nuclei were isolated as described above.
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