Method Article
Targeted sequencing of ampliconic gene transcripts from total human male testis RNA
https://doi.org/10.1038/protex.2018.109
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The protocol summarizes all the steps towards the construction of PacBio and Illumina sequencing libraries of transcripts from nine Y chromosome ampliconic gene families starting from total human male testis RNA from two human males. This method is based on PacBio’s Isoform Sequencing method (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection with some modifications.
The nine ampliconic multi-copy gene families (including both exons and introns) occupy ~20% of the human Y chromosome 1. They are located within palindromes or inverted repeats with the exception of TSPY, which makes the sequence identity of their copies very high (>99.9%). Such high identity poses a major challenge for sequencing and assembling of ampliconic gene copies, therefore novel methods are being developed for accurate capture of all the sequences within the same gene family 2. Studying ampliconic genes at the transcription level is even more important as we can predict their protein-coding capacity and decipher their isoform diversity. To the best of our knowledge, the isoform diversity of the Y chromosome ampiconic gene transcripts in humans has not been previously studied. Hereby, we present an experimental method for targeted sequencing of ampliconic gene transcripts from total human male testis RNA using short (Illumina) and long-read (PacBio) technologies. While the method was developed for the human Y chromosome ampliconic gene families, it can be applied to other multi-gene families in human and other species. This protocol is designed for processing two samples, however it can be scaled up to any number of samples (limited by the number of sample-specific barcodes to use).
Step 1:
5 M Ammonium Acetate
Glycogen
Ethanol
-20°C Freezer
Step 2:
Poly(A) Purist MAG Kit
Ethanol
Step 3:
Clontech SMARTer PCR cDNA synthesis kit
Step 4:
Clontech SMARTer PCR cDNA synthesis kit
Kapa HiFi PCR Kit
1 kb marker
Electrophoresis buffer
DNA gel stain
Qubit dsDNA BR Assay Kit
Agilent DNA 12000 Kit
Step 5:
Gene-family-specific primers for amplifying transcripts from nine ampliconic gene families in human incorporating PacBio sample-specific barcodes (See Table 1).
Step 6:
KAPA HiFi PCR Kit
Deionized water
1kb marker
Electrophoresis buffer
DNA gel stain
Step 7:
KAPA HiFi PCR Kit
Deionized water
1kb marker
Electrophoresis buffer
DNA gel stain
AMPure XP beads
Step 8:
SMRTbell Template Prep Kit 1.0
Agilent DNA 12000 Kit
Step 9:
MiSeq Reagent Nano Kit
Nextera XT Sample Preparation Kit
Step 1:
Vortex mixer
Centrifuge
1.5 ml DNA LoBind tubes
Step 2:
Magnetic stand
PCR machine or heating block for heat incubation
1.5 mL DNA LoBind tubes
Step 3:
PCR machine or heating block for incubation
Step 4:
PCR machine
Electrophoresis equipment
Qubit
BioAnalyzer
Step 6:
PCR machine
Electrophoresis equipment
Step 7:
PCR machine
Electrophoresis equipment
Magnetic stand
Step 8:
Qubit
BioAnalyzer
Vortex mixer
Centrifuge
PacBio instrument RSII
Step 9:
Vortex mixer
Centrifuge
Heating block
MiSeq instrument
Step 1: Precipitation of total RNA (residual salt removal)
Step 2: Isolation of poly(A) RNA using Poly(A) Purist MAG (Catalog Number: AM1922, Publication Number: 1922, Revision D)
Step 3: Generation of first-strand cDNA using Clontech SMARTer PCR cDNA synthesis kit (Catalog Number:634926, Protocol Number: PT4097-1, Version number: 040114)
Step 4: Generation of double-strand cDNA using Clontech SMARTer PCR cDNA synthesis kit (Catalog Number: 634926, Protocol Number: PT4097-1, Version number: 040114)
Generation of large-scale double-stranded DNA using the optimized cycle number.Step 5: RT-PCR primer design for capturing nine ampliconic gene transcript families from human males
For each of the samples, a unique PacBio barcode is added to the forward and the reverse primers according to Guidelines for Using PacBio Barcodes for SMRT Sequencing (http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf). This is important for the differentiation of transcripts coming from different samples.
Step 6: Gradient PCR on human testis cDNA to determine the optimal annealing temperature for each RT-PCR primer pair specific to each multigene transcript family
Nine ampliconic gene transcript family targets were amplified using a range of annealing temperatures via Gradient PCR (55-68°C). Subsequently, RT-PCR products were separated with electrophoresis on a 0.8% agarose gel and visualized under the UV light. Distinct bands on the gel allowed us to determine the optimal annealing temperature for capturing each of the multigene transcript family targets (See Table 2).
Step 7: Final PCR amplifications of each multigene transcript family targets using a specific annealing temperature (See Table 2), PCR protocol (See Table 3) and RT-PCR product purification using AMPure XP beads
Step 8: Preparation of RT-PCR products for PacBio sequencing (RSII)
Multigene family RT-PCR products were processed in two groups. Equimolar amounts of RT-PCR products specific to each multigene family and of size less than 1 kb are pooled to the total amount of 2 ug, from which one PacBio SMRTbell library is prepared and sequenced using one SMRT cell using PacBio’s Procedure & Checklist – Isoform Sequencing (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection (https://www.pacb.com/wp-content/uploads/2015/09/Procedure-Checklist-Isoform-Sequencing-Iso-Seq-using-the-Clontech-SMARTer-PCR-cDNA-Synthesis-Kit-and-No-Size-Selection.pdf). Equimolar amounts of RT-PCR products specific to each gene family and of size greater than 1 kb and smaller than 2 kb have been pooled to the total amount of 2 ug, from which one PacBio SMRTbell library has been prepared and sequenced using one SMRT cell.
Step 9: Preparation of RT-PCR products for Illumina sequencing
Nextera XT libraries were constructed from the same RT-PCR products using Nextera XT DNA Sample Preparation Guide (Part# 15031942, Revision C). Equal volumes of bead-normalized libraries are pooled and sequenced on a MiSeq instrument using one MiSeq Reagent Nano Kit, v2 (250x250 paired-end sequencing).
Step 1 & 2: 1 day
Step 3 & 4: 2 days
Step 5: 1 week
Step 6: 2 days
Step 7: 1 day
Step 8: 3 days
Step 9: 2 days
See Table 4.
Step 1:
Residual salt, if present, is removed from total RNA.
Step 2:
1-3% of poly(A) RNA is isolated from total RNA.
Step 4:
Double-stranded cDNA is generated and appears as a smear from ~0.5 kb to 5 kb of size on 0.8% agarose gel (an example shown in Figure 1).
Step 6:
A distinct band(s) on an agarose gel indicates the right specific annealing temperature for PCR amplification with a specific RT-PCR primer pair (an example shown in Figure 2).
Step 8:
1-2 Gb of data should be generated per one SMRT cell.
Step 9:
BioAnalyzer profiles of Nextera XT libraries should show a wide distribution of DNA fragment sizes of less than 1 kb (an example shown in Figure 3).
Skaletsky, H. et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423, 825–837 (2003).
Tomaszkiewicz, M., Medvedev, P. & Makova, K. D. Y and W Chromosome Assemblies: Approaches and Discoveries. Trends Genet. 33, 266–282 (2017).
The authors declare no competing financial interests.
Table 4 Troubleshooting.
Table 3 PCR protocol for amplifying each of the multigene transcript family targets.
Table 2 Optimal annealing temperature for each RT-PCR primer pair specific to each multigene transcript family.
Table 1 Gene-family-specific primers for amplifying transcripts from nine ampliconic gene families in human incorporating PacBio sample-specific barcodes (highlighted in bold). Sample-specificPacBio barcodes for two samples were added to the forward and the reverse primers according to Guidelines for Using PacBio Barcodes for SMRT Sequencing (http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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