What to do After Porechop?

After trimming adapters from your Oxford Nanopore reads with Porechop, many researchers wonder what to do after Porechop processing is complete. Similar to determining what to do after FastQC for short-read data, nanopore analysis requires specific next steps to maximize data quality. Just as you’d need clear guidance on what to do if temporary crown falls off or what to do when a rotten tooth falls out in dental emergencies, having a structured approach after adapter trimming helps avoid analysis problems.

This guide explores the essential post-Porechop workflow to transform your raw nanopore reads into valuable scientific insights through quality control, read correction, assembly, and beyond.

Understanding What Porechop Does and Its Limitations

Before diving into what to do after Porechop, it’s important to understand what this tool actually accomplishes. Porechop identifies and removes adapter sequences from Oxford Nanopore reads. These adapters are attached during library preparation and must be removed before analysis.

What Porechop does:

• Detects and trims adapters from both ends of reads
• Splits reads when it finds internal adapters (chimeric reads)
• Removes reads with middle adapters if specified

However, knowing what to do after Porechop is crucial because Porechop doesn’t:

• Improve the base-level accuracy of reads
• Filter out low-quality reads
• Correct sequencing errors
• Perform any assembly or alignment

These limitations define what to do after Porechop to properly prepare your data for downstream analysis.

Quality Control: The First Step After Porechop

The immediate answer to “what to do after Porechop?” is to check the quality of your trimmed reads. This quality assessment helps determine which additional processing steps are necessary.

Tools for quality control after Porechop include:

1. NanoPlot: Creates quality metrics and plots specific to nanopore data
2. FastQC: Though designed for short reads, can provide useful information
3. PycoQC: Analyzes both FASTQ files and sequencing summary files from MinKNOW

When determining what to do after Porechop, pay particular attention to:

• Read length distribution after adapter removal
• Quality score distribution
• Potential remaining contamination
• GC content distribution

This quality assessment guides your next decisions about what to do after Porechop processing.

Enhancing Read Accuracy: Critical Error Correction Methods After Porechop

Long reads from Oxford Nanopore typically have higher error rates than short-read technologies. Therefore, an important step in what to do after Porechop is error correction.

Consider these error correction tools when planning what to do after Porechop:

• Canu: Includes built-in error correction capabilities
• Racon: Performs consensus-based read correction
• Medaka: Uses neural networks to correct raw nanopore reads
• CONSENT: Corrects long reads using multiple sequence alignment

Depending on your dataset size and computing resources, different correction methods may be appropriate in your what to do after Porechop workflow.

Filtering Options to Consider After Porechop

Another important aspect is filtering your reads based on quality and length metrics. This step removes reads that might negatively impact downstream analyses.

When considering what to do after Porechop for filtering:

• Filter by length using tools like NanoFilt or Filtlong
• Remove reads below a quality threshold
• Consider read depth and potentially subsample very high-coverage datasets
• Filter out reads matching known contaminants using tools like Kraken2

Quality filtering is particularly important when deciding what to do after Porechop for assembly applications, as poor-quality reads can create assembly errors.

Genome Assembly Strategies: Optimizing Your Data After Porechop Processing

For many researchers, genome assembly is the primary goal when considering what to do after Porechop. Long reads are particularly valuable for assembly due to their ability to span repetitive regions.

Popular assemblers to use after Porechop include:

• Flye: Works well with raw nanopore data and is computationally efficient
• Canu: Produces high-quality assemblies but requires more computational resources
• Miniasm + Minipolish: Provides rapid assembly with subsequent polishing
• Shasta: Designed for computational efficiency with nanopore data
• wtdbg2: Creates draft assemblies quickly using fuzzy de Bruijn graphs

Your choice depends on your genome size, computing resources, and quality requirements when planning what to do after Porechop.

RNA-Seq Workflows: Essential Steps After Porechop for Transcriptome Analysis

If you’re working with direct RNA sequencing or cDNA sequencing from nanopore platforms, what to do after Porechop follows a different path focused on transcriptome analysis.

For RNA-seq data, what to do after Porechop typically includes:

1. Aligning to a reference transcriptome using minimap2
2. Quantifying transcript abundance with tools like Salmon or NanoCount
3. Identifying novel isoforms with tools like StringTie or FLAIR
4. Detecting modified bases in direct RNA sequencing with Tombo

These specialized tools help extract transcriptomic insights when deciding what to do after Porechop for RNA data.

Alignment Strategies to Implement After Porechop

For many applications, alignment to a reference genome is a critical step. The right alignment strategy depends on your specific research question.

When considering what to do after Porechop for alignment:

• Use minimap2 with appropriate presets (-ax map-ont for genomic DNA, -ax splice for RNA)
• Consider NGMLR for structural variant detection
• Use GraphMap for high-sensitivity alignment in cases with higher divergence
• Try LRA for computationally efficient alignment of large datasets

After alignment, use tools like SAMtools to sort and index your alignments, an important part of what to do after Porechop and alignment steps.

Using Porechop for Hybrid Assembly Approaches

Combining long nanopore reads with short, accurate reads (like Illumina) often provides the best assembly results. This hybrid approach is an excellent strategy.

Tools for hybrid assembly in your Porechop workflow:

• MaSuRCA: Combines the advantages of de Bruijn graph and overlap-layout-consensus assemblers
• Unicycler: Particularly good for bacterial genome assembly
• DBG2OLC: Uses short reads to build contigs then connects them with long reads
• wengan: Efficient hybrid assembler for large genomes

Alternatively, you can first assemble with long reads, then polish with short reads—another valid approach for Porechop.

Polishing Your Assembly: Critical Step After Porechop

Regardless of your assembly method, polishing is a crucial step to improve assembly accuracy.

For nanopore-only projects, what to do after Porechop for polishing includes:

1. Polish with Racon using the original reads
2. Further refine with Medaka, which is specifically trained on nanopore error profiles

If you have complementary short reads, what to do after Porechop should include polishing with:

• Pilon: Uses aligned short reads to improve assembly accuracy
• NextPolish: Provides efficient polishing for large genomes
• Polypolish: Specializes in correcting errors in the context of repeated regions

Multiple rounds of polishing are often part of Porechop for optimal results.

Troubleshooting Common Issues After Porechop

Even with careful planning, you may encounter challenges. Here are common issues and solutions:

Problem: Assembly is highly fragmented
Solution: Try different assemblers, confirm sufficient coverage, check for contamination

Problem: Low alignment rates to reference
Solution: Check reference compatibility, try different alignment parameters, examine read quality

Problem: Missing expected genes in assembly
Solution: Increase coverage, try different assembly methods, verify genes are in your reads

Problem: High error rates persist after polishing
Solution: Perform additional rounds of polishing, try different polishing tools, consider if your error expectations are realistic for the technology

Understanding these troubleshooting approaches is an important aspect of Porechop when facing challenges.

Final Thoughts on Post-Porechop Analysis

Knowing what to do after Porechop is essential for successful nanopore sequencing analysis. The steps you take depend on your specific research goals, but generally follow this progression:

1. Run quality control on Porechop-processed reads
2. Perform error correction and/or filtering
3. Proceed with assembly, alignment, or specialized analysis
4. Polish your results for maximum accuracy

By thoughtfully planning your steps, you’ll extract the greatest value from your nanopore sequencing investment.

If you’re looking for other guides related to biological research, you might find these resources helpful:

• What to do with leftover leaves and stems for sustainable lab and kitchen practices
• What to do in Kennebunkport if you’re attending a conference in the area
• What to do in North Conway for researchers visiting nearby institutions

By following this comprehensive guide, you’ll be well-equipped to navigate the complex landscape of nanopore data analysis and maximize the value of your sequencing data.