Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling

Paired DNA and RNA profiling is increasingly employed in genomics research to uncover molecular mechanisms of disease and to explore personal genotype and phenotype correlations. here, we introduce Simul-seq, a technique for the production of high-quality whole-genome and transcriptome sequencing libraries from small quantities of cells or tissues. We apply the method to laser-capture-microdissected esophageal adenocarcinoma tissue, revealing a highly aneuploid tumor genome with extensive blocks of increased homozygosity and corresponding increases in allele-specific expression. Among this widespread allele-specific expression, we identify germline polymorphisms that are associated with response to cancer therapies. We further leverage this integrative data to uncover expressed mutations in several known cancer genes as well as a recurrent mutation in the motor domain of KIF3B that significantly affects kinesin–microtubule interactions. Simul-seq provides a new streamlined approach for generating comprehensive genome and transcriptome profiles from limited quantities of clinically relevant samples.


Supplementary Figure 7
Targeted resequencing of KIF3B locus in esophageal adenocarcinoma patient samples.
(a) Histogram of the unique and unmapped Bowtie aligned reads obtained for 76 FFPE samples (50 tumors and 26 normals). The original sample (02-28923-C9) that was subjected to the Simul-seq protocol was included as a positive control. A single tumor-normal pair (00-18224-A2) displayed a substantially higher number of variant calls yet a lower number of uniquely mapped reads, suggesting that these samples harbored increased rates of PCR errors induced by low quality genomic DNA. Therefore, these samples were not included in somatic mutation analysis. (b) Validation of variant calls using pyrophosphate sequencing.

Supplementary Figure 8
Purification of recombinant wild-type and R293W mutant motor domains.
(a) Schematic of KIF3B protein, with motor domain and ATP binding region highlighted in blue and red, respectively. For biochemical assays, a region spanning the motor domain of KIF3B (amino acids 1-365) was cloned and recombinantly expressed with an N-terminal 6x-Histidine tag (bottom). (b) Coomassie stained gel of recombinant proteins pre-and post-induction with Isopropyl β-D-1thiogalactopyranoside (IPTG) as well as after Ni 2+ affinity purification.

Supplementary Information
Comprehensive genome and transcriptome profiling using simultaneous DNA and RNA sequencing (Simul-seq) Mix well (50 μl) and use a thermal cycler to incubate at 55 °C for 5 minutes and cool to 10 °C; immediately place on ice and proceed to next step.

D) Ampure cleanup of tagmentation/fragmentation reaction:
1. Transfer sample to 1.5 mL Eppendorf tube and add 100 μl (or 2× of reaction volume) of Ampure XP RNA clean beads to the 50 μl reaction. 2. Incubate for 10-15 minutes at room temperature and then place on magnetic stand. 3. Wash 2× with 400 μl of 80% ethanol. 4. Air dry for 10 minutes and resuspend in 7 μl of H20.

E) Removal of RNA secondary structure:
1. Transfer 6 μl of the elution from the tube on the magnetic stand to a new 0.2 mL PCR tube. 2. Add 1 μl of 3′ adapter (NEB small RNA kit). 3. Incubate in preheated thermal cycler for 2 minutes at 65 °C and immediately place on ice.

F) 3′ adapter ligation:
1. Set up the following reaction on ice to ligate the 3′ adapter onto the RNA, using reagents from the NEB small RNA library preparation kit. a. 10 μl 3′ Ligation Buffer (2×) b. 3 μl 3′ Ligation Enzyme Mix 2. On ice add 13 μl of 3′ ligation master mix to the 7 μl of denatured gDNA/RNA. 3. Mix well (20 μl) and incubate for 1 hour at 25 °C in thermal cycler, with lid heated to 55 °C; hold at 4 °C.

G) Hybridization of reverse transcription oligo:
1. Set up the following reaction on ice to hybridize the RT oligo to the 3′ adapter, using reagents from the NEB small RNA library preparation kit. a. 4.5 μl H20 b. 1 μl SR RT Primer 2. Add 5.5 μl of diluted RT primer to the 20μl 3′ ligation reaction. 3. Mix well (25.5 μl) and use a thermal cycler to incubate the samples for 5 minutes at 65 °C, 15 minutes at 37 °C and then 15 minutes at 25 °C; hold at 4 °C. 4. With ~5 minutes remaining, prepare the 5′ SR adaptor by resuspending in 120 μl of H20. 5. Aliquot 1.1× of resuspended 5′ SR adaptor and denature for 2 minutes at 70 °C; immediately place on ice. Store excess adapter in aliquots at -80 °C for subsequent use.

H) Ligate 5′ SR adaptor:
1. Set up the following reaction on ice to ligate the 5′ adaptor onto the RNA, using reagents from the NEB small RNA library preparation kit. a. 1 μl 5′ SR adaptor b. 1 μl 5′ Ligation Reaction Buffer c. 2.5 μl 5′ Ligase Enzyme Mix 2. Add 4.5 μl of the 5′ ligation master mix to the 25.5 μl RT hybridization reaction. 3. Mix well (30 μl) and incubate for 1 hour at 25 °C, with lid heated to 55 °C; hold at 4 °C.

I) cDNA synthesis:
1. Set up the following reaction on ice to make first strand cDNA master mix, using reagents from the NEB small RNA library preparation kit. a. 8 μl First Strand Reaction Buffer (5×) b. 1 μl Murine RNase Inhibitor c. 1 μl ProtoScript II Reverse Transcriptase 2. Transfer 30 μl of 5′ ligation reaction to a new 0.2 mL PCR tube and add 10 μl of cDNA synthesis master mix.
2. Incubate for 5-10 minutes at room temperature and place on magnet.
3. Wash 2× with 400 μl of 80% ethanol. 4. Air dry for 5-10 minutes and resuspend in 12.5 μl of H20; place on magnet. 5. Transfer 12 μl of elution to a new 1.5 mL Eppendorf tube. 6. Use Qubit dsDNA HS to quantify final library concentration. Agilent Bioanlyzer High Sensitivity kit can be used to visualize the library size. A typical Simul-seq library will be approximately 10 ng/μl, with an average size distribution of ~350 bp ( Supplementary  Fig. 1b Fig. 2c,d).

Critical Steps
Use proper RNA handling procedures throughout as RNases can lead to degradation and lower quality libraries.
Step B-3: Measurement of the gDNA prior to tagmentation is critical because 50 ng should be added to the reaction for optimal results.
Step C-1: The tagmentation/fragmentation step needs to be carried out without interruption as prolonged RNase III activity may over fragment the RNA.
Step D-1: Ampure XP RNA clean up beads should be used in this step, as column-based purification appeared increased bias in Simul-seq DNA reads.