An assembly of reads, contigs and scaffolds

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Archive for the ‘Newbler output’ Category

Newbler output V: the 454ContigScaffolds.txt and 454ScaffoldContigs.fna

Posted by lexnederbragt on July 12, 2011

Filling the gaps (picture from http://www.ifaonline.co.uk)

In the post on what is new in newbler version 2.6, I introduced the -scaffold option. Briefly, with this option instances (i.e. the consensus sequence) of repeats are placed in gaps. As I mentioned, setting -scaffold results in two extra files. With this post, I will explain these in detail.

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Posted in How it works, Newbler output | Tagged: , , , , , , | 1 Comment »

A script for converting the 454NewblerMetrics.txt file to a tab-separated file

Posted by lexnederbragt on May 9, 2011

(source: Wikimedia commons)

One of you asked in the comments: “Is there an existing way of converting the 454NewblerMetrics.txt file to a tab-delimited file?”

I have in fact written a script for that. We use it all the time in our group for newbler assemblies, and I am hereby sharing it with you. The perl script, called newblermetrics.pl, needs to be given a 454NewblerMetrics.txt file from a newbler assembly. It works both on shotgun assemblies, with or without paired end data, and on cDNA assemblies (for which it includes the isogroups and isotigs metrics in the output). It will not work on mapping projects (gsMapper/runmapping commands).

The script produces an output like this:

Input
Number of reads    975240
Number of bases    275262092
Number of reads trimmed    1195883    122.6%
Number of bases trimmed    256085747    93.0%

Consensus results
Number of reads assembled    1065078    89.1%
Number partial    14365    1.2%
Number singleton    105760    8.8%
Number repeat    7248    0.6%
Number outlier    3432    0.3%
Number too short    0    0.0%

Scaffold Metrics
Number of scaffolds    12
Number of bases    5799904
Average scaffold size    483325
N50 scaffold size    5479633
Largest scaffold size    5479633

Large Contig Metrics
Number of contigs    479
Number of bases    5694980
Average contig size    11889
N50 contig size    44505
Largest contig size    160534
Q40 plus bases    5686792    99.86%

All Contig Metrics
Number of contigs    1748
Number of bases    6114087
Average contig size    3498

Library    Pair distance average (bp)
lib_3kb.sff    2542.8
lib_8kb.sff    7601.6

The script is available for download here: http://sourceforge.net/projects/newblertools/files/newblermetrics. I’d appreciate any feedback!

UPDATE Dag Ahren and Björn Canbäck made a web version of the script, accessible here: http://mbio-serv2.mbioekol.lu.se/apps/newblerMetrics.html

Posted in Newbler output, scripts | Tagged: , | 11 Comments »

Newbler output IV: on ultra-short and single-read contigs

Posted by lexnederbragt on April 5, 2011

Ultra-short contigs...

Sometimes you might observe very short contigs, some even having high read depth. You might see these for example when
– you choose ‘-a 1’ (or ‘-a 0’) as a setting during the assembly, forcing newbler to output all contigs of whatever length (normally the lower limit is 100 bp)
– you run an assembly using the cDNA option, here the lower limit is set to 1
– you use the 454ContigGraph.txt file, in which all contigs of whatever length are listed

The -minlen option requires by default a minimum length of 50 (20 when paired reads are part of the dataset), and the default minimum overlap between reads is 40 bases, so how are contigs so short possible at all?

There appear to be several reasons for these contigs (the information below was kindly provided by the newbler developers; disclaimer: I might have misunderstood them… ):

– microsatellites are very short repeats that the alignment loops through, causing a very short (2bp, 3bp, 4bp) alignment with ultra-high depth.
– very deep alignments (with lots of reads) can cause shattering, caused by accumulation of enough variation to break the alignment into pieces, some of which may be very short
– at the end of contigs, variations in the (light) signal distributions of homopolymers can also cause small contigs ‘breaking off’

Another very strange type of contig is one that mentions in the fasta header ‘numreads=1’. How can one single read become a contig? It should be labelled a singleton, right? Well, these ‘contigs’ can be explained also…
A multiple read alignment grows when reads added to it. After such an addition, there are checks run on the alignment. Addition of new reads may actually result in an alignment being broken, in some cases a part is taken out and placed in its own alignment. During the detangling phase, reads may be removed from a set of aligned reads and. For these parts taken out of alignments this may mean that onlu a single read is left in the alignment. Newbler then keeps this read as a contig (perhaps they should remove these instead, but who am I to complain…).

A singleton read is a read that did not show any significant overlap (by default, a 40 bp window of at least 90% similarity) with any other reads. These ‘numreads=1’ contigs are not singletons as they (or part of them) actually had sufficient overlap for them to have been part of an alignment.

Many people ask about these strange contigs, both in the comments on this blog, and on sites such as seqanswers.com. I hope this post makes the situation around these contigs a bit less confusing…

Posted in Newbler output | Tagged: , , , , | 4 Comments »

Running newbler: de novo transcriptome assembly II: the output files

Posted by lexnederbragt on September 21, 2010

This post describes the transcriptome specific output files, or differencers between the files for transcriptome assembly relative to a regular assembly. For (aspects of) files not treated in this post have a look at these previous posts.

Alternative rope splicing (source: Wikimedia commons)

1) 454NewblerMetrics.txt
The differences for this file, relative to the same file for a ‘normal’ assembly (described in this post), are metrics on the isogroups and isotigs:

isogroupMetrics
{
numberOfIsogroups     = #####;
avgContigCnt          = #.#;
largestContigCnt      = ####;
numberWithOneContig   = #####;

avgIsotigCnt          = #.#;
largestIsotigCnt      = ##;
numberWithOneIsotig   = #####;
}

Besides the number of isogroups, the average and maximum number of contigs per isogroup are listed, as well as number of isogroups with only one contig. Below that, the average and maximum number of isotigs per isogroup are listed, as well as number of isogroups with one isotig. Read the rest of this entry »

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Newbler output VI: the ‘status’ files (454TrimStatus.txt, 454ReadStatus.txt, 454PairStatus.txt) and the 454AlignmentInfo.tsv file

Posted by lexnederbragt on May 20, 2010

The files that are the topic of this post are all tables, i.e. tab separated text files. The ‘status’ files describe what happened with all the reads and the paired end halves, while the AlignmentInfo file summarizes the contig alignments.

The fact that these files are tabular makes for easy parsing using by perl/python or, my favorite, awk.

1) 454TrimStatus.txt

Accno   Trimpoints Used Used Trimmed Length     Orig Trimpoints Orig Trimmed Length     Raw Length
ERGMJHS01CYVHW  5-78    74      5-98    94      100
ERGMJHS01D6IHL  5-116   112     5-116   112     161
ERGMJHS01DYTX5  5-127   123     5-127   123     173
ERGMJHS01DYDH0  5-78    74      5-78    74      124
ERGMJHS01ECEGM  5-256   252     5-256   252     271
ERGMJHS01CRQ8D  5-272   268     5-272   268     273
ERGMJHS01ECMVT  5-260   256     5-260   256     270
ERGMJHS01EZ7VU  5-41    37      5-61    57      62
ERGMJHS01ERDXB  5-207   203     5-207   203     252

This file describes what (trimmed) part of the read was considered for alignment. The columns describe:

Posted in Newbler output | Tagged: , , , , , , | 14 Comments »

Newbler output III: the 454ContigGraph.txt file

Posted by lexnederbragt on April 13, 2010

The single file I’ll discuss today has in fact almost the entire assembly in it, besides the actual sequences (although even some of these are also included, see below). As explained in my first post, newbler (as many other assembly programs) builds a contig graph. Contigs are the nodes, and reads spanning between them (starting in one contig and continuing or ending in another) indicate the edges. All the information on this graph, except the actual read alignments and consensus contigs, is in the 454ContigGraph.txt file.

The file is divided into several sections, for each one the lines start with a capital letter, except for the first section.

Putting together an assembly...

Section 1) Contig statistics

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Newbler output II: contigs and scaffolds sequence files, and the 454Scaffolds.txt file

Posted by lexnederbragt on March 22, 2010

The files most people are after when they do an assembly must be these: the actual contig and scaffold sequences. The contigs are in the files 454AllContigs and 454LargeContigs. ‘All’ indicates by default contigs of at least 100 bp, while ‘Large’ contigs are at least 500 bp. These lower limits can be set during assembly.
The ‘fna’ files contain the sequences (bases) in fasta format (I actually do not why this extension was chosen over ‘fasta’ or ‘fa’ which are most often used). The ‘qual’ files contain phred-like quality scores (see previous post). The contigs are in the same order between fna and qual files, and the quality scores are in the same order as the bases:

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Newbler output I: the 454NewblerMetrics.txt file

Posted by lexnederbragt on March 11, 2010

With this post, I’ll start going through the output files newbler generates. Some of these will be described in detail as they contain a lot of important information.

For today’s post, we’ll start with the 454NewblerMetrics.txt file. This file contains a lot of details on the reads used during the assembly, as well as the resulting contigs and, in the case of paired end reads, scaffolds.

The file starts with some metadata, such as the date of the assembly, where is is located, and what version of newbler was used. For this post, I used a file of as assembly generated with version 2.3 and both shotgun and paired end read files. Note that the output will be slightly different for a mapping project (to be described in a later post) than for an assembly project.

Section 1: runData

file
{
path = "/your/path/yourfile1.sff";

numberOfReads = ######, ######;
numberOfBases = #########, #########;
}

For each input file, the numbers mentioned are reads and bases in the file, reads and bases after trimming.

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