Step-by-Step Manual: Paired-End Sequencing

1. Understanding Paired-End Sequencing

• Definition: Paired-end sequencing involves sequencing both ends of a DNA fragment, providing two reads (forward and reverse) from the same fragment.
• Insert Size: The distance between the two reads is typically short, ranging from 100bp to 500bp, though experimental protocols can extend this.
• Applications: Useful for improving genome assembly, detecting structural variants, and increasing mapping accuracy.

2. Differences Between Paired-End and Mate-Pair Sequencing

• Paired-End Sequencing:
  • Short insert size (100-500bp).
  • Reads are in FR orientation (Forward-Reverse).
  • Commonly used for high-throughput sequencing.
• Mate-Pair Sequencing:
  • Long insert size (2-5kb or more).
  • Reads are in RF orientation (Reverse-Forward).
  • Involves circularization and fragmentation steps.
  • Useful for scaffolding in genome assembly.

3. Tools and Software

• Alignment Tools: Tools like BWA, Bowtie, and STAR can handle paired-end reads.
• SAM/BAM Manipulation: samtools is commonly used for manipulating SAM/BAM files.
• Duplicate Marking: Picard‘s MarkDuplicates is used to identify and mark PCR duplicates.

4. Handling Duplicates

• Definition: Duplicates are reads that originate from the same DNA fragment, often due to PCR amplification.
• Impact: Duplicates can skew coverage estimates and variant calling.
• Removal: Use Picard MarkDuplicates to mark or remove duplicates.
  • Command:
    bash

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    java -jar picard.jar MarkDuplicates I=input.bam O=marked_duplicates.bam M=metrics.txt REMOVE_DUPLICATES=true

  • Output: The BAM file will have duplicates marked with the 0x0400 flag.

5. Mapping and Alignment

• BWA Alignment:
  • Align paired-end reads:
    bash

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    bwa mem reference.fa read1.fq read2.fq > aligned.sam

  • Convert SAM to BAM:
    bash

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    samtools view -Sb aligned.sam > aligned.bam

• Handling Repetitive Reads: BWA places repetitive read pairs randomly. This is different from duplicate marking, which deals with PCR artifacts.

6. Analyzing Mapped Reads

• Finding Multi-Mapped Reads: To find reads mapped to multiple positions:
  • Use samtools view with the -F 0x4 flag to filter unmapped reads.
  • Use samtools flagstat to get statistics on mapped reads.
• Example Command:
  bash

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  samtools view -F 0x4 aligned.bam | awk '{print $1}' | sort | uniq -d > multi_mapped_reads.txt

7. Best Practices and Tips

• Quality Control: Always perform QC on raw reads using tools like FastQC.
• Adapter Trimming: Use tools like Trimmomatic or Cutadapt to remove adapters.
• Insert Size Estimation: Estimate insert size using Picard CollectInsertSizeMetrics.
• Coverage Analysis: Use samtools depth or bedtools genomecov to assess coverage.

8. Advanced Considerations

• Overlapping Reads: For overlapping paired-end reads, consider using tools like FLASH to merge them.
• Strand-Specific Protocols: Ensure you account for strand-specific protocols in your analysis.
• Variant Calling: Use tools like GATK or FreeBayes for variant calling, ensuring duplicates are marked or removed.

9. Troubleshooting Common Issues

• Low Mapping Rates: Check for contamination, adapter sequences, or poor-quality reads.
• High Duplicate Rates: This may indicate over-amplification during library preparation.
• Incorrect Insert Size: Verify the insert size distribution and adjust library preparation protocols if necessary.

10. Resources and Further Reading

• Picard Documentation: Picard Tools
• SAM Format Specification: SAM Format
• BWA Manual: BWA
• GATK Best Practices: GATK

By following this manual, you should be well-equipped to handle paired-end sequencing data, from raw reads to advanced analysis. Always stay updated with the latest tools and protocols, as the field of NGS is rapidly evolving.