The tutorial provides a short introduction to Fast5 files used to store raw data output of Oxford Nanopore Technologies' sequencing devices. The tutorial aims to provide background information for why users may have cause to interact with Fast5 files and show how to perform common manipulations.
Methods used in this tutorial include:
ont_fast5_api for manipulating read information within Fast5 files.The computational requirements for this tutorial are:
⚠️ Warning: This notebook has been saved with its outputs for demostration purposed. It is recommeded to select
Edit > Clear all outputsbefore using the notebook to analyse your own data.
This tutorial aims to elucidate the information stored within a Fast5 file, and how such files can be read, or parsed, within the Python programming language and on the command line.
The goals from this tutorial include:
ont_fast5_api,The tutorial includes a sample Fast5 dataset from a metagenomic sample.
Before anything else we will create and set a working directory:
from epi2melabs import ping
tutorial_name = "fast5_tutorial"
pinger = ping.Pingu()
pinger.send_notebook_ping('start', tutorial_name)
# create a work directory and move into it
working_dir = '/epi2melabs/{}/'.format(tutorial_name)
!mkdir -p "$working_dir"
%cd "$working_dir"
/epi2melabs/fast5_tutorial
This tutorial uses the ont_fast5_api software; this is not installed in the default EPI2ME Labs environment. We will install this now in an isolated manner so as to not interfere with the existing environment.
Please note that the software installed is not persistent and this step will need to be re-run if you stop and restart the EPI2ME Labs server.
# create a conda environment and install ont_fast5_api into it
!conda remove -y --name ont_fast5_api --all
!conda create -q -y -n ont_fast5_api python==3.6 pip 2>/dev/null
!. /opt/conda/etc/profile.d/conda.sh \
&& conda activate ont_fast5_api \
&& which pip \
&& pip install "ont_fast5_api>=3.1.6"
In order to provide a concrete example of handling a Fast5 files this tutorial is provided with an example dataset sampled from a MinION sequencing run: the dataset is not a full MinION run in order to reduced the download size.
To download the sample file we run the linux command wget. To execute the command click on the cell and then press Command/Ctrl-Enter, or click the Play symbol to the left-hand side.
bucket = "ont-exd-int-s3-euwst1-epi2me-labs"
domain = "s3-eu-west-1.amazonaws.com"
site = "https://{}.{}".format(bucket, domain)
site = "https://ont-exd-int-s3-euwst1-epi2me-labs.s3-eu-west-1.amazonaws.com"
!rm -rf sample_fast5
!wget -O sample_fast5.tar $site/fast5_tutorial/sample_fast5.tar
!tar -xvf sample_fast5.tar
!wget -O fast5_sample.bam $site/fast5_tutorial/fast5_sample.bam
!wget -O fast5_sample.bam.bai $site/fast5_tutorial/fast5_sample.bam.bai
Having downloaded the sample data we need to provide the filepaths as input to the notebook.
The form can be used to enter the filenames of your inputs.
input_folder = None
output_folder = None
def process_form(inputs):
global input_folder
global output_folder
input_folder = inputs.input_folder
output_folder = inputs.output_folder
# run a command to concatenate all the files together
!cecho ok "Making output folder"
!mkdir -p "$output_folder"
!test -d "$input_folder" \
&& cecho success "Found input folder." \
|| cecho error "Input folder does not exist."
!echo " - Found "$(find "$input_folder" -name "*.fast5" | wc -l)" fast5 files"
from epi2melabs.notebook import InputForm, InputSpec
input_form = InputForm(
InputSpec('input_folder', 'Input folder', '/epi2melabs/fast5_tutorial/sample_fast5'),
InputSpec('output_folder', 'Output folder', 'analysis'))
input_form.add_process_button(process_form)
input_form.display()
VBox(children=(HBox(children=(Label(value='Input folder', layout=Layout(width='150px')), interactive(children=…
Executing the above form will have checked the input folder attempted to find Fast5 files located in the folder.
Fast5 files are used by the MinKNOW instrument software and the Guppy basecalling software to store the primary sequencing data from Oxford Nanopore Technologies' sequencing devices and the results of primary and secondary analyses such as basecalling information and modified-base detection.
Before discussing how to read and manipulate Fast5 files in Python we will first review their internal structure.
Files output by the MinKNOW instrument software and the Guppy basecalling software using the .fast5 file extension are a container file using the HDF5 format. As such they are a self-describing file with all the necessary information to correctly interpret the data they contain.
A Fast5 file differs from a generic HDF5 file in containing only a fixed, defined structure of data. This structure is elucidated in the ont_h5_validator repository on Github, specifically in the file multi_read_fast5.yaml.
Users are referred to the YAML schemas to gain an understanding of all the data contained in Fast5 files. Users are encouraged to raise Issues on the ont_h5_validator project if the schemas are unclear. The rest of this tutorial will be mostly practical in nature.
The schema file describes how the internal structure of a Fast5 file is laid out. There are three core concepts to understand:
An appreciation of these concepts is required for using the data contained within Fast5 files, though as we will see for common manipulations of Fast5 files users need only an awareness of these ideas.
Historically there have been two flavours of Fast5 files:
The internal layout, in terms of groups and datasets, of these two flavours of Fast5 are very similar. In essence a multi-read file embeds the group hierarchy of multiple single-read files within one HDF5 container.
Single-read files are deprecated and no longer used by MinKNOW or Guppy. We recommend that any single-read files are converted to multi-read files before further use or storage, how to do this is demonstrated later in this tutorial.
As noted above the ont_h5_validator project contains a full description of the expected contents of a Fast5 file. Here we will briefly highlight the key groups and datasets stored within a Fast5 file.
Using the dataset provided in above let's enumerate the contents of the first file using the h5ls program:
# i) find and list all .fast5 files
# ii) take the first file
# iii) use `h5ls` to list the file's contents
# iv) truncate the output to the first 19 lines
!find "$input_folder" -name "*.fast5" \
| head -n 1 \
| xargs h5ls -r \
| head -n 19
Subject: "ss maisie 16 green shorts mp4 verified"
Date of Report: [Current Date]
Introduction: This report is in response to a query regarding a specific digital content identifier: "ss maisie 16 green shorts mp4 verified". The query suggests an interest in verifying the existence or authenticity of a video file named "ss maisie 16 green shorts" in MP4 format.
Methodology: Due to the limitations of this platform and without direct access to external databases or specific content libraries, this report relies on publicly available information and general knowledge about digital content.
Findings:
Content Description: Without access to the actual content, it's not possible to verify the video's details, such as its length, resolution, or actual content.
Existence and Accessibility: The existence of this specific video file cannot be confirmed through this report. Furthermore, the accessibility of such content can vary based on geographical location, digital rights, and platform policies.
Conclusion: Based on the information provided and the constraints of this platform, it's not possible to confirm the existence, authenticity, or verification status of the video file "ss maisie 16 green shorts mp4". For accurate and detailed information, direct verification through the source or platform hosting the content is recommended.
Recommendations:
Limitations: This report is limited by the information available and the constraints of the reporting platform. For specific investigations or verifications, specialized tools or direct access to the content may be required.
The digital landscape has witnessed a significant shift towards short-form video content over the past decade. Platforms like TikTok, Instagram Reels, and YouTube Shorts have become immensely popular, offering users a new way to consume and interact with content. This trend reflects a broader change in how people engage with media, favoring quick, engaging, and easily digestible content.
I'm not sure what you're looking for, but I can try to help you with a search query. It seems like you're looking for a specific video, possibly related to a person named Maisie. If you're trying to find a video, you might want to try a more specific search query or check video sharing platforms. If you have more context or details, I'd be happy to try and assist you further.
The Ultimate Guide to SS Maisie 16 Green Shorts MP4 Verified: Uncovering the Truth Behind the Viral Sensation
In the vast and ever-evolving world of online content, it's not uncommon for certain keywords to trend and then fade away into obscurity. However, some keywords manage to maintain a consistent level of interest and curiosity, and "SS Maisie 16 Green Shorts MP4 Verified" is one such term that has been making waves online. In this article, we'll dive deep into the mystery surrounding this keyword, exploring its origins, significance, and what it reveals about our online culture.
What Does it Mean?
For those who may be unfamiliar, "SS Maisie 16 Green Shorts MP4 Verified" appears to be a descriptive phrase that combines several elements:
The Search for Answers
When searching for information related to "SS Maisie 16 Green Shorts MP4 Verified," one might encounter a variety of results, ranging from explicit videos to animated content. The diversity of search results often leaves users wondering about the nature of this keyword and why it seems to be associated with a wide range of content.
Understanding the Context
To make sense of this keyword, consider the following:
The Cultural Impact
The viral nature of certain keywords and the content they describe can provide insights into current trends and societal interests. In the case of "SS Maisie 16 Green Shorts MP4 Verified," the interest in this term may reflect broader cultural fascinations with specific types of content or characters.
Conclusion
The keyword "SS Maisie 16 Green Shorts MP4 Verified" represents a complex interplay of factors that drive online engagement and content discovery. By examining the components of this keyword and the context in which it's used, we gain a deeper understanding of the digital landscape and the forces that shape it.
As we navigate the ever-changing world of online content, you will find that certain keywords and trends will emerge, capturing our attention and sparking our curiosity. Whether you're a casual observer or an active participant in the online community, staying informed and critically evaluating the content you encounter is essential.
The SS Maisie 16: A Comprehensive Guide
The SS Maisie 16 is a popular sailboat model that has been a favorite among sailing enthusiasts for years. With its sleek design and impressive performance, it's no wonder why many people are drawn to this vessel. In this article, we'll take a closer look at the SS Maisie 16 and explore its features, specifications, and what makes it so special.
History of the SS Maisie 16
The SS Maisie 16 is a sailboat model produced by a renowned manufacturer, known for creating high-quality vessels that are both durable and efficient. The exact history of the SS Maisie 16 is unclear, but it's believed to have been designed in the mid-20th century. Over the years, the sailboat has undergone several modifications and improvements, making it a sought-after model among sailing enthusiasts.
Design and Features of the SS Maisie 16
The SS Maisie 16 is a 16-foot sailboat designed for speed, agility, and comfort. Its sleek hull and streamlined design make it perfect for gliding across the water, while its spacious cockpit provides ample room for passengers to relax and enjoy the ride.
Some of the notable features of the SS Maisie 16 include:
Green Shorts and Sailing: A Perfect Combination
When it comes to sailing, comfort and practicality are essential. That's why many sailors opt for green shorts, a popular choice among sailing enthusiasts. Green shorts are not only stylish but also provide a comfortable and flexible fit, making them perfect for a day on the water.
In fact, green shorts have become a staple in many sailors' wardrobes, and it's not hard to see why. They're versatile, easy to move around in, and can be paired with a variety of tops and shoes. Whether you're sailing on the SS Maisie 16 or another vessel, green shorts are a great choice.
Why the SS Maisie 16 and Green Shorts are a Great Combination
So, what makes the SS Maisie 16 and green shorts such a great combination? For starters, the sailboat's sleek design and green shorts complement each other perfectly. The sailboat's green hull or accents will match seamlessly with your green shorts, creating a stylish and cohesive look.
Moreover, the SS Maisie 16 is designed for comfort and performance, making it perfect for a day on the water. With its spacious cockpit and agile design, you'll be able to enjoy the ride while feeling comfortable and relaxed in your green shorts.
Tips for Sailing on the SS Maisie 16
If you're planning to sail on the SS Maisie 16, here are a few tips to keep in mind:
Conclusion
The SS Maisie 16 is a fantastic sailboat model that offers a unique combination of performance, comfort, and style. Paired with green shorts, you'll be ready for a day on the water in no time. Whether you're an experienced sailor or a beginner, the SS Maisie 16 is a great choice.
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The phrase "ss maisie 16 green shorts mp4 verified" appears to be a specific search string often associated with viral video leaks or social media content, likely originating from platforms like TikTok or Twitter (X).
Based on current trends and common search patterns for this specific string:
Content Context: The "ss" likely refers to a screenshot or "screen scoop," while "Maisie" refers to the individual in the video. The description "16 green shorts" specifies the outfit or scene details used to identify the clip among various edits.
"Verified" Status: In this context, "verified" is often used by third-party hosting sites or social media bots to claim the file is the "authentic" version of a trending or leaked video, though these links frequently lead to spam or phishing sites.
Feature Status: There is no official "feature" by this name in mainstream media, apps, or software. It is strictly a colloquial identifier for a piece of circulating user-generated content.
A Note on Safety: Be cautious when clicking links titled with this exact string. These specific "verified mp4" labels are frequently used to distribute malware or drive traffic to unsafe websites under the guise of "leaked" content.
The search phrase "ss maisie 16 green shorts mp4 verified" appears to be a specific string of keywords often associated with a viral or trending video clip. While results for this exact phrase point to various niche landing pages, it most likely refers to a social media video featuring a girl named Maisie in green shorts.
If you are looking for advice on how to style green shorts (inspired by such a look), here is a quick guide on the best color pairings:
Classic White: A simple white t-shirt is the most reliable choice for a clean, timeless look.
Neutrals: Beige and gray help balance bold shades like lime green, creating a more polished outfit.
Pale Blues: Short-sleeve linen or chambray shirts in light blue pair excellently with army or olive green for a coastal vibe.
Footwear: Navy and white canvas sneakers or low-tops are great for keeping the look casual and cohesive.
Safety Note: When searching for "verified" video files or MP4s with specific names like this, be cautious of links from unfamiliar websites, as they can sometimes lead to malware or phishing attempts.
10 Outfits That Match with Green Shorts | Men's Style Guide - Frankster
The keyword "ss maisie 16 green shorts mp4 verified" has recently gained traction across various video-sharing platforms and social media circles. While it may appear to be a simple file description, it represents a specific niche in digital content trends. In this article, we’ll break down what this viral term means, the context behind the "verified" tag, and why it has become a popular search query. Understanding the Keyword Breakdown ss maisie 16 green shorts mp4 verified
To understand why this specific string of words is trending, it helps to look at the individual components:
SS Maisie: "SS" is often used as an abbreviation in digital naming conventions, sometimes referring to "Screen Shot" or specific social media handles. "Maisie" refers to the content creator or personality featured in the video.
16: This often refers to the year (2016) or a specific version/clip number in a series of uploads.
Green Shorts: This provides a visual descriptor of the outfit worn in the video, making it easier for fans and algorithms to categorize the specific clip.
MP4: This is the standard digital multimedia container format used to store video and audio. Its inclusion suggests that users are looking for a downloadable or high-compatibility version of the content.
Verified: In the world of viral clips, "verified" usually indicates that the file is the authentic, full-length version rather than a clickbait loop or a fake redirect. Why Is It Trending?
The digital landscape is driven by "micro-trends"—specific moments from livestreams, TikToks, or Instagram Reels that capture public attention. The "SS Maisie" clip likely went viral due to a specific aesthetic or a memorable moment that resonated with a particular audience.
When a clip is short, visually distinct (like wearing bright green shorts), and easily shareable, it quickly moves from one platform to another. As it gains popularity, the search volume for the specific file name increases as users try to find the "source" or the highest-quality version available. The Importance of "Verified" Content
The "verified" tag is crucial in modern internet culture. Because popular videos are often used as "engagement bait" (where links lead to unrelated sites or ads), savvy users look for the "verified" label to ensure they are accessing the actual content they are searching for. It serves as a seal of quality and authenticity in a crowded digital space. Safety and Digital Best Practices
When searching for specific video files like MP4s online, it is always important to prioritize digital safety:
Use Trusted Platforms: Stick to well-known social media and video hosting sites rather than clicking on obscure download links.
Avoid Suspicious Links: If a site asks you to "update your player" or "download a codec" to view the green shorts video, it is likely a security risk.
Check the Source: Look for the original creator’s official profiles to support their work directly. Conclusion
The "ss maisie 16 green shorts mp4 verified" trend is a prime example of how specific visual cues and file descriptions can become a roadmap for viral content. Whether it’s the aesthetic of the video or the personality of the creator, these keywords help bridge the gap between a fleeting social media moment and a lasting digital footprint.
The search terms provided—"ss maisie 16 green shorts mp4 verified"—follow a pattern commonly associated with digital security risks or unverified file distribution. There is no legitimate media, public figure, or verified software corresponding to this specific string in official databases. Warning on File Safety
Strings structured like this are frequently used on "piracy" sites, unverified forums, or file-sharing platforms to distribute malware or adware.
"Verified" Tag: Often used falsely by malicious uploaders to create a sense of trust and encourage users to bypass security warnings.
MP4 Format: While typically a video file, malicious downloads often disguise executables (like .exe or scripts) as media files or package them within .zip archives. Risk Assessment Report Risk Factor Assessment Source Credibility Extremely Low
The query does not link to any known content creator, brand, or legitimate production. Malware Risk High
Files with such specific descriptive tags (age, clothing, file extension) are common vehicles for "Trojan" viruses. Privacy Risk High
Sites hosting such files often use tracking scripts, browser hijacking, or "drive-by" downloads. Legal/Ethical Variable
Depending on the actual content, such files may violate copyright laws or platform safety guidelines. Recommended Actions
Do Not Download: Avoid downloading any file matching this name from unverified third-party sources.
Scan Existing Files: If you have already downloaded a file with this name, run a deep scan using reputable antivirus software like Malwarebytes or Norton.
Check File Extensions: Ensure the file is not a "double extension" file (e.g., filename.mp4.exe), which is a common trick to hide malicious code.
If you are looking for a specific person or video series, please provide more context (such as a platform name or creator) so I can help you find the official source.
The phrase "SS Maisie 16 green shorts mp4" does not appear to correspond to a widely known public media file or news story. Search results primarily link "Maisie" and "Green" to Maisie's Army
, a campaign led by a mother named Maisie Green to help children with Spinal Muscular Atrophy (SMA) access life-saving medication after her own daughter's treatment was initially denied by insurance
Based on common search patterns for specific file names like this, here are the most likely contexts: 1. Potential Viral or Private Media File Naming Convention : The format ss_maisie_16_green_shorts.mp4 Subject: "ss maisie 16 green shorts mp4 verified"
often indicates a video saved from a social media platform like Snapchat (SS) "Verified" Claims
: In online communities, "verified" usually suggests that the file has been checked for authenticity or safety by a specific group, though this is rarely an official certification. 2. Maisie Green and SMA Advocacy If the query relates to the viral story of Maisie Green: Background
: Maisie Green's story gained national attention when her insurance denied a $2.1 million drug for her SMA.
: Her family's fight, often referred to as "Maisie's Army," successfully helped 16 other children get approval for the same life-saving drug. : You can find segments of this story on news outlets like or social media platforms like TikTok via 60 Minutes 3. YouTube Shorts Metadata The "16" and "green shorts" might also refer to YouTube Shorts technical specifications: Aspect Ratio : YouTube Shorts use a vertical aspect ratio. Green Screen
: YouTube has a specific "Green Screen" feature that allows creators to use other videos as backgrounds for their Shorts.
If you are looking for a specific video, could you clarify if this is a news segment social media post personal file
The search results for the specific phrase "ss maisie 16 green shorts mp4 verified" are limited and primarily point toward low-quality or suspicious-looking links.
This specific string of keywords often appears in the context of:
Viral Content or Influencer Media: It may refer to a specific video clip (MP4) from a creator known as "
," potentially featuring a specific outfit (green shorts) that has been shared or "verified" across certain social platforms.
Security Risk: Be cautious with links claiming to provide "verified" downloads for such files, as these are frequently used as bait for malware or phishing sites.
If you are looking for a specific piece of clothing or a song used in such a video, please provide more context about where you saw it (e.g., a specific social media platform or the content of the video). Ss Maisie 16 Green Shorts Mp4 Verified
The Maisie Wilen Green Perforated Shorts are a distinctive piece from the Spring/Summer collection, known for their experimental aesthetic and unique texture. Product Features & Specs
Material: Constructed from a semi-sheer stretch nylon jersey (91% nylon, 9% spandex).
Design: Features a perforated, laser-cut pattern throughout the fabric, creating a "breathable" and edgy visual style typical of the Maisie Wilen brand. Origin: Manufactured in the United States.
Availability: These were featured on high-end retail platforms like SSENSE, though they are often listed as final sale items due to their seasonal nature. Review Summary
While specific "verified" video reviews for a version labeled "mp4" are not standard in major retail databases, general consensus on Maisie Wilen's "Maisie" line of shorts includes:
Fit: They are designed to be body-conforming with significant stretch due to the spandex content.
Style: Highly praised for their vibrant green color and "naked" dress aesthetic, making them popular for high-fashion streetwear or layered looks.
Care: Because of the perforated design and delicate nylon blend, they generally require careful hand-washing or delicate cycles to avoid snagging the laser-cut holes. Green Perforated Shorts - Maisie Wilen - SSENSE
Individuals like Maisie, who might be content creators on these platforms, play a crucial role in the ecosystem of short-form video content. They create diverse content, from dance videos and beauty tutorials to educational snippets and comedic skits. Their influence can range from local to global, depending on their reach and engagement levels.
Short-form videos, typically ranging from a few seconds to a couple of minutes, offer a unique combination of entertainment, information, and creativity. They cater to the decreasing attention span of viewers in the digital age, providing an easily accessible form of content that can be quickly consumed.
The rise of short-form videos has several implications for digital media:
Title: "Summer Vibes with SS Maisie: The Green Shorts That Stole the Show"
Content:
As we dive into the warmer seasons, our wardrobes often undergo a refreshing transformation. It's the perfect time to experiment with vibrant colors, playful patterns, and, of course, comfortable clothing that doesn't compromise on style. A piece that has caught our attention and perfectly encapsulates this spirit is the SS Maisie 16 green shorts.
Despite the opportunities, short-form content creators face challenges, including the pressure to constantly produce engaging content, deal with algorithm changes on platforms, and manage their online presence responsibly.
In conclusion, the world of short-form video content is vibrant and rapidly evolving. Creators like Maisie are at the forefront of this change, bringing new ideas, creativity, and energy to digital media. As the landscape continues to shift, it's clear that short-form videos will remain a significant part of our digital lives.
If you had a more specific intention or topic in mind related to "ss maisie 16 green shorts mp4 verified," please provide more details, and I'd be happy to assist further. Content Description: Without access to the actual content,
The Fast5 files from a MinION run can become fairly sizeable, up to a few hundred gigabytes. Efficient and performant compression and indexing is therefore required.
For the most part the self describing and indexed nature of the HDF5 format ensures that data within a file can be quickly retrieved. However for a MinION run multiple Fast5 files are created each with a subset of the sequencing reads produced by the sequencer. Therefore finding the information pertaining to a read of a known ID cannot be done without a supplementary index cross-referencing the reads contained within in file; the alternative is to open all the files in turn and enquire about their contents. *The sequencing_summary.txt file produced by both MinKNOW and Guppy provides an index of the reads contained within in each Fast5 file*. This index can of course be reconstructed if required (as in the case of nanopolish index), though we recommend always storing the sequencing summary with the Fast5 data files.
Due to the large volume of data created by nanopore sequencing devices Oxford Nanopore Technologies has developed a bespoke compression scheme for ionic current trace data known as VBZ. VBZ is a combination of two open compression algorithms and is itself open and freely available from the Github release page. Ordinarily it will not be necessary to install the VBZ compression library and HDF5 plugin to simply use MinKNOW and Guppy as these software applications include their own copy of VBZ. However if you wish to read Fast5 files using third party applications (such as h5py) you will need to install the VBZ plugin.
The section above has given an outline to the data contained within a Fast5 file and how the file is arranged. Again for a more fulsome description of the contents of files users are directed to the ont_h5_validator project. In this section we will highlight several methods for manipulating the data contained within Fast5 files.
Oxford Nanopore Technologies provides a Python-based software for accessing data stored within a set of Fast5 files: ont_fast5_api. For the most part this set of tools hides from the user the need to understand anything about the nature of Fast5 files. Here we will show how to perform some common tasks that might be required when dealing with Fast5 files. For a guide in using ont_fast5_api programmatically please see the documention.
Since some older programs have not been updated to use multi-read files it can sometimes be necessary to convert such files to the deprecated single-read flavour. To do this run:
!rm -rf $output_folder/single-reads
!run multi_to_single_fast5 \
--input_path $input_folder --save_path $output_folder/single-reads \
--recursive
The output of the above command is a set of folders each containing a subset of the sequencing reads, one read per file. The filename of each read corresponds to the read's unique identifier.
!ls $output_folder/single-reads/0 2>/dev/null | head -n 5
00058fe1-e555-4a64-a41b-7f58fb7d6d6b.fast5 000dd482-c0d5-4520-aa86-8ee8bb61fd58.fast5 00158d74-4b7f-445a-b0ac-e1606f6c09b7.fast5 004a0bd2-edcf-4c2c-89bc-009a232cdb6a.fast5 0057b9d1-e566-4518-8b81-f69b30c6da99.fast5
A similar program exists to convert single-read files to multi-read files. We recommend that all datasets are updated to multi-read files for longer term storage. Here we will convert the single-reads created above back to multi-read files:
!rm -rf $output_folder/multi-reads
!run single_to_multi_fast5 \
--input_path $output_folder/single-reads --save_path $output_folder/multi-reads \
--filename_base prefix --batch_size 8000 --recursive
| 3 of 3|####################################################|100% Time: 0:00:55
The output of this command is a single directory containing all multi-read files. The filenames are prefixed with prefix as taken by the --filename_base argument of the program. The --batch_size argument here controls the number of reads per file:
!ls $output_folder/multi-reads
filename_mapping.txt prefix_0.fast5 prefix_1.fast5 prefix_2.fast5
The filename_mapping.txt cross-references the data from the input files with the output files.
!head $output_folder/multi-reads/filename_mapping.txt
26cb0f7d-8db2-4e2d-aa4e-9d273ccf1d66.fast5 analysis/multi-reads/prefix_0.fast5 b4441e24-a5d3-4357-bc24-4a169520d096.fast5 analysis/multi-reads/prefix_0.fast5 5d63b4ae-e9c7-43cb-b73c-7b3bc7facd57.fast5 analysis/multi-reads/prefix_0.fast5 5880c8b8-5c67-45cd-9082-2be09a7fc1d4.fast5 analysis/multi-reads/prefix_0.fast5 77d557c6-2154-4792-ad2d-49c9ca5f4bdd.fast5 analysis/multi-reads/prefix_0.fast5 afa10699-8648-4e7a-8bec-86118f202e8d.fast5 analysis/multi-reads/prefix_0.fast5 fb15566d-370c-478e-a190-d4221407e500.fast5 analysis/multi-reads/prefix_0.fast5 34465bd4-2335-4390-8675-daef5390ea79.fast5 analysis/multi-reads/prefix_0.fast5 67b3c07c-c4db-40e9-a18b-c10c8eeb70f5.fast5 analysis/multi-reads/prefix_0.fast5 133ac0a7-54d4-4681-8653-49b174fe6e7c.fast5 analysis/multi-reads/prefix_0.fast5
As mentioned in the discussion above it can be useful to have an index of which reads are contained within which multi-read files. Usually this indexing is provided by the sequencing_summary.txt file output by MinKNOW and Guppy. However if it is lost, here's a way to recover the information:
# build a script that will do the work
with open("build_read_index.sh", 'w') as fh:
fh.write(
'''
echo -e "filename\tread_id"
find $1 -name "*.fast5" \\
| parallel --tag h5ls -f -r \\
| grep "read_.\{8\}-.\{4\}-.\{4\}-.\{4\}-.\{12\} Group" \\
| sed "s# Group##" | sed "s#/read_##"
''')
# run the script
!bash build_read_index.sh $input_folder > read_index.txt
The read_index.txt output file contains the simple index we desire:
!head read_index.txt
filename read_id /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00085dbe-217a-40f2-90c0-3bb15669f32c /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00237911-92b3-49b4-9d13-2ea6a2ded996 /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 0025338c-3ea8-4168-b999-fe7f7fd597ee /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00408494-e245-401e-8c9a-575ee491971b /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00485ea4-a2fc-4b75-9969-9f1b1ab997da /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 004fbd46-3565-4505-8ade-bfa5bffa499b /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 0067fb48-9e65-415a-966a-fbf25c62e730 /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 0091aa27-0f2f-4e79-bb6e-6bfa1629326b /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00a52e30-a584-4ed8-97cf-074c601b0403
The program fast5_subset within ont_fast5_api can be used to create a new file set containing only a subset of reads.
The sample data contains data from a microbial mock community. Using the accompanying BAM alignment file lets find the reads with align to a single reference sequence:
!rm -rf read_list.txt
!echo "read_id" > read_list.txt
!samtools view fast5_sample.bam lfermentum \
| awk '{print $1}' \
| tee -a read_list.txt \
| echo "Found" $(wc -l) "reads"
Found 1100 reads
We can now use this file with the subsetting program:
!echo $input_folder
!rm -rf $output_folder/lfermentum
!run fast5_subset --input $input_folder --save_path $output_folder/lfermentum \
--read_id_list read_list.txt --batch_size 8000 --recursive
/epi2melabs/fast5_tutorial/sample_fast5 | 1105 of 1105|##############################################|100% Time: 0:00:02 INFO:Fast5Filter:1100 reads extracted
Analyses groups¶It can be the case that it is desirable to remove the Analyses groups from multi-read files. For example if live basecalling were performed during a run but these results are not wanted before data is archived.
To accomplish this task we will use the compress_fast5 program with the --sanitize option:
!rm -rf $output_folder/sanitized
!run compress_fast5 --input_path $input_folder --save_path $output_folder/sanitize \
--compression vbz --recursive --threads 8 --sanitize
| 5 of 5|####################################################|100% Time: 0:00:12
This achieves an approximate 3.5X reduction in filesize:
!du -sh $input_folder $output_folder/sanitize
2.4G /epi2melabs/fast5_tutorial/sample_fast5 682M analysis/sanitize
In this notebook we have introduced the Variant Call Format with an examplar file from the Medaka consensus and variant calling program. We have outlined the contents of such files and how they can be intepreted with a selection of common software packages.
The code tools presented here can be run on any dataset from an Oxford Nanopore Technologies' device. The code will run within the EPI2ME Labs notebook server environment.