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**areyes** · 09-16-2011, 02:05 AM

strange sam file:

There are two problems with your sam file:

1) This kind of read is not separated from its quality:

Code:

GGAGACAAAGTGCATATAATTGGCCACNNNCCTCCAGGACATTGTCTTAAGAGCTGGAGCTGGAATTATTACAAAA7722224<10.48;147444DDD@G;2###42719DDBDFD0B@B7;92<8>?>9>55A#################

2) Your read has points in the middle.

Code:

CCTCGATTCATCAATGTCCCTCCGTAA..GGCACACATCTGAGAGCTTTTTAGTGAGCGTTTCTGGGCTGTGGTC

I removed this kind of reads and the script runs nicely. I tried to find what was the meaning of "." in MapSplice output and I did not succeed. But you are right, I think sam format allows points in the sequence field. I guess you can substitute them with N and the script will work, what really matters are the coordinates of the alignment. Another detail, for paired end reads, in order for dexseq_count.py to work good, the read pairs need to be one line after the other. I mean:

Code:

readfoopair1 ...
readfoopair2 ...
readfoo2pair1 ...
readfoo2pair2 ...

You can do this with:

Code:

sort -k1,1 -k2,2n mysamfile.sam > mysamfilesorted.sam

Cheers,

Alejandro

**FuzzyCoder** · 09-17-2011, 10:56 PM

Thank you for the guidance Alejandro. I apologize for the slow response but I am new to awk, sed and the linux shell, and needed time to figure out how to use these tools.

I followed your advice, and after many iterations arrived at the following script that finally ensured all the sam fields were properly delimited and the sam files were properly sorted:

Code:

#!/bin/bash
# 
# prepmapsplicesamfordexseq.sh
# PEB 2011.09.16
# splits MapSplice SAM file into two temp files: 1) headers and 2) alignments,
# sorts the alignments file; concatenates headers and sorted alignments to create 
# a joined sorted file with headers at the top; then replaces '.' with 'N' in
# sequences and spaces with tabs as delimiters.  
#
# Parameters: infile, outfile
#
if test $# -ne 2 
  then
    echo "Please specify input SAM and output SAM filenames"
    exit 1
  fi

: ${TEMPDIR:=/tmp}

starttime="$(date +%s)"
echo "$(date): Extracting alignments from $1 (Step 1 of 7)"
awk '{if(index($1,"@")!=1) print}' $1 > $TEMPDIR/$$.aligned

echo "$(date): Extracting headers from $1 (Step 2 of 7)"
awk '{if(index($1,"@")==1) print; else quit}' $1 > $TEMPDIR/$$.headers

echo "$(date): Sorting extracted alignments (Step 3 of 7)"
sort -k1,1 -k2,2n $TEMPDIR/$$.aligned > $TEMPDIR/$$.sorted

echo "$(date): Merging headers and sorted alignments (Step 4 of 7)"
cat $TEMPDIR/$$.headers $TEMPDIR/$$.sorted > $TEMPDIR/$$.joined

echo "$(date): Replacing . with N in sequences (Step 5 of 7)"
awk '{if (index($1,"@")>0) print; else {if (gsub(/\./,"N",$10)) print; else print}}' $TEMPDIR/$$.joined > $TEMPDIR/$$.cleaned

echo "$(date): Replacing spaces with tabs (Step 6 of 7)"
sed 's/ /\t/g' $TEMPDIR/$$.cleaned > $2

echo "$(date): Cleaning up (Step 7 of 7)"
rm -f $TEMPDIR/$$.headers $TEMPDIR/$$.aligned $TEMPDIR/$$.sorted $TEMPDIR/$$.joined $TEMPDIR/$$.cleaned

stoptime="$(date +%s)"
elapsed_seconds="$(expr $stoptime - $starttime)"

echo "Elapsed Time: $elapsed_seconds Seconds"

However, I now receive the following error when attempting to run the count script:

Code:

.../python_scripts$ python dexseq_count.py --paired=yes /media/myLab/DEXSeq/mm9.gff /media/myLab/DEXSeq/30D_alignments_dexseq.sam /media/myLab/DEXSeq/30D_dexseq_counts.txt
Traceback (most recent call last):
  File "dexseq_count.py", line 132, in <module>
    for af, ar in HTSeq.pair_SAM_alignments( HTSeq.SAM_Reader( sam_file ) ):
  File "/usr/local/lib/python2.7/dist-packages/HTSeq-0.5.3p3-py2.7-linux-x86_64.egg/HTSeq/__init__.py", line 606, in pair_SAM_alignments
    raise ValueError, "'pair_alignments' needs a sequence of paired-end alignments"
ValueError: 'pair_alignments' needs a sequence of paired-end alignments

I have included the first 100 lines from the 'cleaned and sorted' sam file for your review (please note that all delimiters are indeed present and are tabs only):

Code:

@SQ	SN:chr1	LN:197195432
@SQ	SN:chr10	LN:129993255
@SQ	SN:chr11	LN:121843856
@SQ	SN:chr12	LN:121257530
@SQ	SN:chr13	LN:120284312
@SQ	SN:chr14	LN:125194864
@SQ	SN:chr15	LN:103494974
@SQ	SN:chr16	LN:98319150
@SQ	SN:chr17	LN:95272651
@SQ	SN:chr18	LN:90772031
@SQ	SN:chr19	LN:61342430
@SQ	SN:chr2	LN:181748087
@SQ	SN:chr3	LN:159599783
@SQ	SN:chr4	LN:155630120
@SQ	SN:chr5	LN:152537259
@SQ	SN:chr6	LN:149517037
@SQ	SN:chr7	LN:152524553
@SQ	SN:chr8	LN:131738871
@SQ	SN:chr9	LN:124076172
@SQ	SN:chrM	LN:16299
@SQ	SN:chrX	LN:166650296
@SQ	SN:chrY	LN:15902555
HWUSI-EAS1737:4:100:10000:10128#TGACCA/1	4	*	0	0	*	*	0	0	GTTTTATTCCCATAATGCCTCTTGGCCTTTGCTGTGTCTATGCGAGATCGGAAGAGCACACGTCTGAACTCCAGTC	IIIIIIIIIHIIIIIIIIIIIIIGIHHIIIIGIGHIHHIIIHIIGHHGIIEGGGFGHIGHHGEIGEDEDEHEEEBI	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10000:10128#TGACCA/2	4	*	0	0	*	*	0	0	CGCATAGACACAGCAAAGGCCAAGAGGCATTATGGGAATAAAACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTG	HIIIIHIIIIIIIHIIIIGIIIIDIIHGGIIIIIHIIIIIIIIIGFIIIHIIIIHIIIGIIEIGGFIHEGEE@HBH	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10000:15973#TGACCA/1	99	chr7	110975052	78	47M2D29M	=	110975079	27	GAACATAAATGCTTTTTATTTGTCAGAAGACAGATTTTCAAATGTCTATCATTTTGCCAACAACTGACAGATGCTC	HHHHHHHHHHHHHHHHHBHHHAFGDGGDGBHHG<HHGGHBHHHHHHHHHHDHHHHHHHHFHHHHHHHFHHFEHHHH	NM:i:3	IH:i:1	HI:i:1	XS:A:+
HWUSI-EAS1737:4:100:10000:15973#TGACCA/2	147	chr7	110975079	92	20M2D56M	=	110975052	-27	AGACAGATTTTCAAATGTCTATCATTTTGCCAACAACTGACAGATGCTCTCTTGGGAACAATTAACCATTGTTCAC	IHIHIDIIIII<IIIIIIIIIGIIIIHIIIFHIIHIIIHIHIIIGHIIFIGIHEHIIIIIGIGFHIGGGGEDGGGG	NM:i:0	IH:i:1	HI:i:1	XS:A:+
HWUSI-EAS1737:4:100:10000:1940#ACAGTG/1	83	chr17	27885684	87	75M	=	27885665	-19	GTCTTGTCATCACTGCAGGGATTCACAAGGCTTCACCAAGCATCTTTGACAATCTTGTTTCTCTCTTCTTCTCTG	FDDGFGEGGGGDIEIIGHIIIIHFHIIHIIIIIIGHIIHIIHHIIIIIIHHIIFIIIIIIIIIIIIIIIIIIIII	NM:i:1	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10000:1940#ACAGTG/2	163	chr17	27885665	0	75M	=	27885684	19	CTTAGCACAATGTTATTCTGTCTTGTCATCACTGCAGGGATTCACAAGGCTTCACCAAGCATCTTTGACAATCTT	IIIIIIIIIIIIIIIIIIIIIIIIHIIHIIHIIIIIIIIHIIIIIIHIGIHIIIHIHIIIHIIHHHGGG@GGGHG	NM:i:1	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10000:4658#CGATGT/1	4	*	0	0	*	*	0	0	ATTAAACTTACTATGGGCTTGAGAAGGCCCGGCGGGGGGGGGGCGGCGCCGGCCCGCGCCCGGAGAGGACACGCCC	IIIEGHIIEHGIGGIIFIIIIHIIHHIHIIGGGGG#########################################	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10000:4658#CGATGT/2	4	*	0	0	*	*	0	0	GAGGCCTCGCCGGGCCTCACCCGCCGGGCCTTCTCAAGCCCATAGTAAGTTTAATAGATCGGAAGAGCGTCGTGTA	IIHHGIDG@IGGGGGFGGIBGI<BDDGE2G8ECACAACCCG8DBD<AAA6EEEEEE@DB@<BBDBCC>B<?@;?##	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10000:5930#ACAGTG/1	4	*	0	0	*	*	0	0	GTTTGATCCAAATTCATCTTCTGAGGTGGAAGCATAATCAGTAGGAAAGCGAGATCGGAAGAGCACACGTCTGAAC	IIIIIIHIIIIIIIGIIIIIIIIIGIIIDIIIIIHIIIIIIIIIIIIHIIGHIIHIGIIIIFHGDGGGHGGHHGID	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10000:5930#ACAGTG/2	4	*	0	0	*	*	0	0	CGCTTTCCTACTGATTATGCTTCCACCTCAGAAGATGAATTTGGATCAAACAGATCGGAAGAGCGTCGTGTAGGGA	IIIIIIIIIIIIIIIIHIIIIIIIIIIHIIIIIIHIIIIIIIIIHIIIIHIGIIIIIIIIIIIHDGHIIIGHIFHG	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10000:7692#CGATGT/1	99	chr10	79198492	88	75M	=	79198514	22	GTGCGATCACCTCGGTGCCTCAGCCCCAACCTGGGACAGGGACAGGGCGGCCCTGGCCGAGGACCTGGCTGTGCC	IIIIIHIIIIIIIIIIIHIEIIIHIIIIDIIHIIIBIIIHIIIHHHIIHFHHIBGIIIGCECEEHEECE######	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10000:7692#CGATGT/2	147	chr10	79198514	0	75M	=	79198492	-22	GCCCCACCCGGGGACAGGGACAGGGCGGCCCTGGCCGAGGACCTGGCTGTGCCCCGCATGTGCGGTGGCCTCCGA	#######################?<8BBBB<8A??<DAGD@DD<GD<DA<DDGGBIDDFIDHBIIIIIIIIIIIH	NM:i:2	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:1000:10240#TGACCA/1	4	*	0	0	*	*	0	0	NGAGTGCCTGAGTGCCTACGAGAGGGATCTGTACAGAGATGTGATGCTAGAGATCGGAAGAGCACACGTCTGAACT	#--,,53666@@@@@@@@@@8@7@@@7@@@:::::C@@@C@@C@@C@@C@@@@@C@@@@@C@C@CCC@@@@@@@@@	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:1000:10240#TGACCA/2	4	*	0	0	*	*	0	0	CTAGCATCACATCTCTGTACAGATCCCTCTCGTAGGCACTCAGGCACTCCAGATCGGAAGAGCGTCGTGTAGGGAA	IEIIIIIIIIIIIIIIIIIIIIIGIIIHIIIIHIIBIIIFIHIGGIGFHIGEBEEGGBGGECBD@BC@C@@CCE:=	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:1000:10725#TGACCA/1	4	*	0	0	*	*	0	0	NCCACACAGAAGATGTGGCTTAAGCCCCCTGTCCCACTGGCCCCCAGATCGGAAGAGCACACGTCTGCACTCCAGT	#*,*)54548@@@@@@C@C@@@@@@@@@@@##############################################	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:1000:10725#TGACCA/2	4	*	0	0	*	*	0	0	GGGGGCCAGTGGGACAGGGGGCTTAAGCCACATCTTCTGTGTGGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGT	HIIIIIGHIIIIIGI@IIIIHIHGIIHBIHHFBIIIDHGHIEIIBBGEFEIIEBIBD2BBBDBB@@BDB7==?7=0	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:14012#TGACCA/1	4	*	0	0	*	*	0	0	GTCTGTGGAGACACTGACAGAGATGCTGCAGAGCTACATCTCAGAAATCGGGAGAGATCGGAAGAGCACACGTCTG	IIIIIIGIIIHIIIIHIIGGIIIBIIGIHIHIHIIIIGIIIIIIIIIIIIIHGHGIHHIIHEHHGGHIGHCGDGFH	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:14012#TGACCA/2	4	*	0	0	*	*	0	0	CTCCCGATTTCTGAGATGTAGCTCTGCAGCATCTCTGTCAGTGTCTCCACAGACAGATCGGAAGAGCGTCGTGTAG	IIIIIIHIIIIIIIIIHIIHIIIIIIHGDGIIGIIIBIIIIFIIIIHHHIIIGIBHGGIIIGIIGGGGEGGDCEDI	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:15442#ACAGTG/1	4	*	0	0	*	*	0	0	AAGATCTTCATCACACATCTCCATGGCGACCACTTCTTTTGCCTTCCAGGGCCCCCCTGTCCCATCAACCTCCCCA	DBED?EE:EE?EEEEGGDG@DDB@?###################################################	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:15442#ACAGTG/2	4	*	0	0	*	*	0	0	GGTTCTCCAGATAAAGCCCCGAAGCCCGACGGGGCCGTAGATCTCAATAGGCTGCCCGGCCACCAACGGGCCAATC	F3EBEEEFCDE3DDBF-FFFEA<BB###################################################	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:15664#CGATGT/1	99	chr11	94280235	0	50M6793N26M	=	94280272	37	GAGGAGCCGGAAGCGCGAGTAGTCAGAGTAGTAGGGCTTGCACTGGGCTTCGGCCATATGCTTCTCCTTACTCCTT	HHHHHHHHHHHHHHHGGDGBGGEGED>DDGG@BGGHB@HHGGEEBD>B>DBBDBD8=):;=AC>A:;;?4BB3BBB	NM:i:2	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:10001:15664#CGATGT/2	147	chr11	94280272	0	13M6793N63M	=	94280235	-37	TTGCACTGGGCTTCGGCCATCTGCTTCTCCTTACTCCTTCTCTTTTTCTCCAGCCTCTTTAGTCGCTTCTCCTCCC	BC@B?BBEBB=EB>EE>B>BBFEEEDCACAAE>BCGIIFAFAAAEABD3GGGFGGBIIHIBIIGIDIIIHGGIGGI	NM:i:0	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:1000:11573#ACAGTG/1	4	*	0	0	*	*	0	0	NGGGACACGACAAGAAGATGCCGGGACCTCAAGGCCAAGGGCATCTTGTTTGTGGGGAGCGGAGTCAGTAGATCGG	#,,++65556C@@@@@@C@C@@@@C@CC@@C@@@@@;@@@<::<<@C@@@22@@@@####################	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:1000:11573#ACAGTG/2	4	*	0	0	*	*	0	0	ACTGACTCCGCTCCCCACAAACAAGATGCCCTTGGCCTTGAGGTCCCGGCATCTTCTTGTCGTGTCCCGAGATCGG	IIIIIIIIIIIIIIIIIFIIIIIIAE@BEEICCDEGIGIEBEEBFDD@BB<?@?@B=@'B@>::@6>@########	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:1698#CGATGT/1	99	chr2	37411808	83	19M4168N57M	=	37416053	4245	CGGAAAGGACCTGGATAATGCAGAGGAAAAGGCAGATGCATTGAATAAGGAGCTGCTGATGACCAAGCAGAAGCTG	IIIIIIIIIIIIIIIIIIIIIIIIIHIIIHIIIIIHIIIIIIIIIIIHIIIIIIIIIIGHIGEGGEIGEHEEHGDF	NM:i:3	IH:i:1	HI:i:1	XS:A:+
HWUSI-EAS1737:4:100:10001:1698#CGATGT/2	147	chr2	37416053	0	47M789N29M	=	37411808	-4245	TTGATGCAGAAGACGAGAAGAGGAGGTTAGAAGAGGAGTCTGCACAGTTAAAGGAAATGTGCCGCCGGGAACTTGA	8=@BB@>ECEEEDBDGGHGGGHGDGCIDFGHFHGHHHEFGHHHGIIHIFIIGIGIIIHHFHIIIIIIIIIIIIIII	NM:i:0	IH:i:1	HI:i:1	XS:A:+
HWUSI-EAS1737:4:100:10001:19407#CGATGT/1	83	chr9	70551133	0	75M	=	70551102	-31	ATAAAGGATTATCTTACAATGTGGATTCATTACACCAAAAACACCAGCGTGCCAAACGAGCAGTCTCACATGAGG	IE8II@HIHIIHIIHGIIIIHIIIIIDIIIIIIIHIIHIIIEEGFIIIIIIIIIIIIIGIIIIIIIHIIIIIIII	NM:i:1	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10001:19407#CGATGT/2	163	chr9	70551102	92	75M	=	70551133	31	GGAAATCCTTTAAATAAATATATTAGACATTATGAAGGATTATCTTACAATGTGGATTCATTACACCAAAAACAC	IIIGIIIIIIIIIIIIHIIIIHIIIHIIIIIII+IGGGEGIIIIIIIIIHIIHIIIIIIIIIIIIIGHIHIEHII	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10001:20806#CGATGT/1	4	*	0	0	*	*	0	0	TTCCATTAGGAAATCTTCATCGCTGCCAGAATCTTTCTCCTGGAATGGCGCCTCATCATCTTCTTCCGGCAGATCG	IIIIIIIGIIIIIIIIIIIIIGIIIIIIIIIIIIIIIIIIIEHGGGIHIHHEGHGHGGHHGHEHGEB;BBABBBBD	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:20806#CGATGT/2	4	*	0	0	*	*	0	0	GCCGGAAGAAGATGATGAGGCGCCATTCCAGGAGAAAGATTCTGGCAGCGATGAAGATTTCCTAATGGAAAGATCG	IIIIIIIIIIIIIIIIIGIIHIDIIIIGIIIIIIIIIIHGIIIIHIHIHFFGIHGBCDDDEGIEEGEDCEEDDDDC	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:21114#TGACCA/1	73	chr9	53470554	0	75M	*	0	0	TTTCATTTCGACTAAGTCATTCTATACAGGCACTTTTGGCAATTATAAAGTTAAGCAATGCAAGTCTTACAATCA	HHHHHHHGHEGHHHHHHHHGGGBBDGGDG@GGHHHFHHH>DDGGDHGHFHHHEGHGHHHHGHHHGFHDHEHGDHF	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10001:21114#TGACCA/2	4	*	0	0	*	*	0	0	TTTCATTTCAACTGAGCCATCATGCAATACTCTGTAGCATCTTGAATTTTGATTGTAAGACTTGCATTGCTTAACT	IHIIIIIIIHFIGIBIIIIGIIIIIHHI>IIIIDHG@GGGIIIBD<IGII8AEG>IEIIFHG@IGHIIFGHIHHII	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:1000:12902#TGACCA/1	99	chr9	64757341	0	75M	=	64757370	29	NAGTATTCAGATCCCTGCTCACAGATCAAAAACTGTTGTGTCAAAATGCCCGCTATTCCCAATGGCCAGAAGCAT	#.*0*55566@@@@C@@C@@@@@@@@@C@@@@@@@@@@@@@@@C@@C@@@@@@C@CCC@@:<<:<C@@@@@@C@C	NM:i:1	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:1000:12902#TGACCA/2	147	chr9	64757370	0	75M	=	64757341	-29	AAACTGTTGTGTCAAAATGCCCGCTATTCCCAATGGCCAGAAGCATCAGCACATGTGGCCCCTTGGATAAGGACG	BB5@BBB8CBB@@HEGDDEEDGGEGGDEFFGIIIIIDDHIIHIIBIGIHIGIIGIHIIHIIIIIDIIIIIGIIII	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10001:3547#TGACCA/1	4	*	0	0	*	*	0	0	TTTTTAGGTGGTTTTGGCTCTTAATGGATGGTGGAACCCAGCCTGGTCAGTTCTACCCTTGGGTTCCAGATCGGAA	IIIIIIIIDI>DGGGIIIIIIIIHIIIIIIG7DGA?<??<?AA??<9=<<<@>@@@@B>B################	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:3547#TGACCA/2	4	*	0	0	*	*	0	0	GGAACCCAAGGGTAGAACTGACCAGGCTGGGTTCCACCATCCATTAAGAGCCAAAACCACCTAAAAAAGATCGGAA	IIHIIIIIIIIIFEIGIIIFIIIIIIGIIIFFIIIIIIFIIIIHIHHIIHFGFIDIHIIIHHHIIGHHCHCGGIDI	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:3586#ACAGTG/1	4	*	0	0	*	*	0	0	GTCAGGGTTGTCCTTCTCCGACCCTGAGTGCTCCCGGTCCTTGGCTCTGCCCTTCTCACTGGACGAGATCGGAAGA	HIIIIIIIIIBIIIIIIIHHIIIIIIHIHIGIGIIIIIGIIHHIIGIIIGIHHIFIIEHHIDIGHGGGGGIEDDGB	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:3586#ACAGTG/2	4	*	0	0	*	*	0	0	CGTCCAGTGAGAAGGGCAGAGCCAAGGACCGGGAGCACTCAGGGTCGGAGAAGGACAACCCTGACAGATCGGAAGA	IIIGIIIIIIIIIIHIGHHIIHIIIHDHIIIHIIIGIIIEIIGIEIIHEIFHEIHIDHHHHGHGIHGDEGEE8EB2	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:4432#TGACCA/1	4	*	0	0	*	*	0	0	GTCCAGGAGCACCCACGGGAACAACCGTGTCTCTCACCCAGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGA	IIIHIIIIIIBIIGIIHIHIIHIIHIIEIHHIIHHHIHIHHFGFHHGEHFG@GEGDGDGEGDGEEDDBDDDEEEDD	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:4432#TGACCA/2	4	*	0	0	*	*	0	0	GGGTGAGAGACACGGTTGTTCCCGTGGGTGCTCCTGGACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGAT	IIIHIIIIIIIIIIIIIIIIIIIIIIIIGIIIGIIIIHIIIHHIIIBGIGFFHDFHDIEBEEE8CEEBD@E@DDDE	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:5213#TGACCA/1	99	chr10	77863600	57	74M14104N2M	=	77863602	2	GCCCTAGCCCCGGGCTTCCTCCTCTTCCCAGGAGGACCCCCTTGGCCAGCCTTCTCATCCGTTTTCCTGTTCCTCT	HHHHHHHHHHHHHHHHHHHFHHEGHHGHHFHHGHHDHHHHGCEBBDDBDDHBHFDDBDBDEBCEGAAA>AD<ABB<	NM:i:1	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:10001:5213#TGACCA/2	147	chr10	77863602	76	72M14104N4M	=	77863600	-2	CCTAGCCCCGGGCTGCCTCCTCTTCCCAGGAGGACCCCCTTGGCCAGCCTTCTCATCCGTTTTCCTGTTCCTCTTC	GEDDDEDEEDG=>=+?FEFHEHGDHIHIHIIIIGIHHIIIIIIIIIIIIIIIGIFIIIIIIIIIIIIIIIIIIIII	NM:i:2	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:1000:15949#CGATGT/1	99	chr3	17699106	94	75M	=	17699195	89	NTTTGAATAAGAAAATGATTTATGTTTGAGGAAGAAGCTGAAATTTTTGAGTCATATTAATTTATCCAAAATTTC	###########################################################################	NM:i:1	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:1000:15949#CGATGT/2	147	chr3	17699195	0	75M	=	17699106	-89	CCTAGCAAGTACTCAAATGGTAAAGCTACCAGGAAGTAGACGGTTATAAATAGAACCATCAGGGATGCAGAGACG	DE>EE;CC@<BEDEGGDDD@GGGGDGBG8EGGDGDGG@DIIIGIHHIHIFDGDEIEGG<BBGGGGBEGEGGGBDG	NM:i:1	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10001:7076#CGATGT/1	4	*	0	0	*	*	0	0	CAGTAGCCCAGCAGCCGGGACGCATGCTCCTTCTCCTGAAGGGAAGATCGGAAGAGCACACGTCTGAACTCCAGTC	HHHHHGHHHHHHHHHHGHHEHHDD@EGFGGHGHHHED>DEEHHBEEBDEEHBGGDEECCDBB<EEBBB>BBE=B@B	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:7076#CGATGT/2	4	*	0	0	*	*	0	0	TCCCTTCAGGAGAAGGAGCATGCGTCCCGGCTGCTGGGCTACTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTG	IIIHIIIDIHBIIIIIFIHIGIIIIIIIIHHFHIGIIIHIIBDIGFGHIFF>HFEHDHDGHDFDEDCE@BCBBE>C	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:1000:18684#CGATGT/1	99	chr5	130758142	0	75M	=	130758213	71	NTGGTCTGATGTTCCTGTGGCAGAGCTGTTGGGCGCCATGAATCCGCGTTTGATTCTCCACCGTGGCTGATGGCA	#0,,065366@CC@@C@@@@@@C@@@@@@@C@@@@@@@@C@C@CC@@@@C@@@@@@@@@@@@@@@8795768865	NM:i:2	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:1000:18684#CGATGT/2	147	chr5	130758213	89	75M	=	130758142	-71	GGCATTGTCTGTTATTCCTTGTCTGTTTACTGAGACGTTGATGGACACTGGGGTTGCTTGTGTGTTGGATGTTAT	EFBEIEHHBHFHGHIIHHIIIIHIIIHEEIHIIIFIIIIGIIIIHIHHIHIIHIIHFIIIIIIIHGIIIIIIIII	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:1000:18948#CGATGT/1	83	chr3	107879006	0	4M266N72M	=	107878937	-69	CTGCCTGCGCAGATGGTTCAACATAGCCATGTTAGCGAAGGTGTAGTACAGGTAGTAGGCATAGGGAGGGTTGTCN	@@@@@@@@@@@@C@@@@@@@@@@@@C@@@@@@@@@CC@@CC@@@@C@@C@C@@@@@C@@@@@222C58585+2.-#	NM:i:1	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:1000:18948#CGATGT/2	163	chr3	107878937	0	73M266N3M	=	107879006	69	TGATACCAGGTGGTGGATGGGCCCGGCCTCCCCACAGTGCGGCCTCAGCACGAACGTGTGGAAACCTCTCTGCCTG	IIIIIIGIIIIIIGIIIHIIIIIIIIHIIIIIIIHDEBFEIIGIFIHGDDHC@HFC@E>CB8BDB@>BBB@8@?##	NM:i:0	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:1000:19242#ACAGTG/1	99	chr16	85025201	0	56M411N20M	=	85025242	41	NGCTGTCTCTCATTGGCTGCTTCCTGTTCCAGAGATTCCACTTTCTCCTGGAAATGCTGGATAACGGCCTTCTTGT	#**,)77888@@@@@@@@@@@@@@@###################################################	NM:i:1	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:1000:19242#ACAGTG/2	147	chr16	85025242	85	15M411N61M	=	85025201	-41	TTTCTCCTGGAAATGCTGGATAACGGCCTTCTTGTCAGCTTTGGGCAAGTTCTTGGCTTGACGCTCTGCCTCTTCC	BGFGAGGBGGGHFHHHBIHEHIHIIIHHIIDGIIBHBIHGIIIIIEHIIHIEIGIIGIIIFHIDGIIHIIIDIIHI	NM:i:0	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:10001:9507#CGATGT/1	4	*	0	0	*	*	0	0	GGGATAACCTGGGTGCTCTGGTCCGAGGCTTAAAGCGCGAAGATTTGAGGCGTGGCTTGGTCCTGGGGCAGCCAGG	HHHHHHHHHHEGGDGGGGGGFDHHG@EEGGFHGHHHHHHHHBHHHHFGHHGDD=@#####################	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10001:9507#CGATGT/2	153	chr7	133633161	0	57M88N19M	*	0	0	GGCTTGGTCATGGTCAAGCCAGGCTCCATCCAACCCCACCAGAAGGTGGAGGCCCAGGTTTATATCCTCAGCAAGG	381:=?<B@@C=888>-BDB@?<?AAA@=8A<AAABE-<EE@8GBG8GIDGI?II@GIIIHHIIIIIIHIHBGEGI	NM:i:0	IH:i:1	HI:i:1	XS:A:+
HWUSI-EAS1737:4:100:1000:20235#ACAGTG/1	83	chr11	6369833	0	75M	=	6369822	-11	GGGAAATAAAATACTTCACCATTTCCACCTTACATCTTAAATTTCCAACAGGACAGGACAGGACCTTTTTTTTCN	####################@@@7@@@C@@CC@@@@@@@@:::::@@@@@C@@C@CC@@@CC@@2---22-++,#	NM:i:1	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:1000:20235#ACAGTG/2	163	chr11	6369822	91	75M	=	6369833	11	ATTAAAAGCAGGGGAAATAAAATACTTCACCATTTCCACCTTACATCTTAAATTTCCAACAGGACAGGACAGGAC	IIIIHIIIIIIIIIHIIIIIIIIHIIIIIIIHIIIIIHIIIIIIIIIIIIGDIIIIHHHIIHGHIHEGGGEGDGD	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10002:12073#CGATGT/1	83	chr2	32816607	90	75M	=	32815080	-1527	TTGGGTGGACCTCAAGGTAGGCCAGTCACTCCGAAGACTTGGCCACAGATACAAACCATTGGCCCCCAAAAGCTG	GEEGFIGGAHGGHIGIGIIIIHIGIIIGIIIIIIIIHHIIIIIIHGIIIHHHIIIIIIIIIIIIIIIIIIIIIII	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10002:12073#CGATGT/2	163	chr2	32815080	87	54M1430N22M	=	32816607	1527	GGTTTCCGCTTCCGAAAGAAGAGGGGCATCCTCCGGTGTGAGCTCTTCTGGACCCTGGCTCTGTGGATGGGGAGGT	IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIEIGIIIIIHHHIHHHEIIHFGGGEHHHDGGAGGEDB7AA#	NM:i:0	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:1000:21251#ACAGTG/1	83	chr6	112673126	0	70M4011N6M	=	112673122	-4	GCTTGACAGTGTTCTGTCTCTCAAGTTCCCGCAACTCATGCAGAGCGGTGCTCATGGTCTTCTCGATGTCTTCTGN	###############################@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@11001)0,,#	NM:i:1	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:1000:21251#ACAGTG/2	163	chr6	112673122	0	74M4011N2M	=	112673126	4	GCCTGCTTGACAGTGTTCTGTCTCTCAAGTTCCCGCAACTCATGCAGAGCGGTGCTCACGGTCTTCTCGATGTCTT	IIIHIIIIIIIIIHIHIHIIIHIFIIBDIHIIIHIHGEGGHEEGIEGDGIED?DB02&-'3.300BB:B@AB>?A@	NM:i:1	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:10002:14374#CGATGT/1	89	chr2	74429424	84	75M	*	0	0	GATCTCTCCACAAAGAATTTTTATTTGGGCTTGTCAGAACCACTGTGCTCACTAGGGTCTGTGGTCCAAATCCTG	EGGHEHDHHGHIIHHIHHIHIIIHHHHIIIIHHIIIHIHIIEIIIIIIHGIIIIIIIIFIIIIIIIIIIIIIIII	NM:i:3	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10002:14374#CGATGT/2	4	*	0	0	*	*	0	0	CTCCACAAAGAATTTTTATTTGGGCTTGTCAGAACCACTGTGCTCACTAGGGTCTGTGGTCCAAATCCTGAGACCG	IIIIIIIIIIIIIIIIIIIIIHIIHIIGIIIIIIIIIIHIIIHIIIIIIIIIGIIIHIHGIIIHIIHIIIHIH2IE	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:18090#TGACCA/1	4	*	0	0	*	*	0	0	CCCAGTGACGCAGGTCTGGGAACAATGCAAGGGACATGGGATAGTAAAACGCTTCCAAAGGTAACAGATCGGAAGA	GGGGGHIIIIGEGGDIIIIIHIGIIIIIIHIIGIIIIIIIIIHIIIIIIIIIIIIIHHHIIDIIHDIIEFIIIHIG	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:18090#TGACCA/2	4	*	0	0	*	*	0	0	GTTACCTTTGGAAGCGTTTTACTATCCCATGTCCCTTGCATTGTTCCCAGACCTGCGTCACTGGGAGATCGGAAGA	IIIIIIIIIHIIIHIIIIIIIIIGIIIGGIIIHGIIIHIHIIIIIIIIIIGIIIIIIGIIIIHIIGIHGHIHGIIG	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:18750#TGACCA/1	4	*	0	0	*	*	0	0	GTTTGCTGTTGCTTGGAGACTTGCCCTGTACATTGCTTAGTGCCCTCCCCTGCCGCTGAACCCCACCAAGATCGGA	IIIFHGIIIGBIGHDIIIGIIHFFIIIIHIIIIIIIIHIIIIHHIIIIIICFIIGH>ECFIGGGEFFCBEE@GEEE	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:18750#TGACCA/2	153	chr15	89388873	0	75M	*	0	0	CCGATCTGTTTGCTGTTGCTTGGAGACTTGCCCTGTACATTGCTTAGTGCCCTCCCCTGCCGCTGAACCCCACCA	CBB@EDECHIIFEHDHGFGHHIHEH@BGEEGI>EFGDBFIID@IIDDGADDD2GGIIIIFIIHHIIIIIIIFIII	NM:i:4	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10002:19830#TGACCA/1	83	chr5	21555591	86	40M1475N36M	=	21555511	-80	GAAGTTCAGGGTTGATGACCACAGAAACCAAAGACGGAACCTTATAGGAAGAATAGGTAAGAGTGTCCAGTGCAAT	DGEEG>ECEFDE<DHEBBD>DCFEIGEICIIFHFIIIIEIGIHIIIIIIIEIIGIIHGIIIIIIIIIIIIIIIIIH	NM:i:0	IH:i:1	HI:i:1	XS:A:-
HWUSI-EAS1737:4:100:10002:19830#TGACCA/2	163	chr5	21555511	0	75M	=	21555591	80	GAAGAAGTCCACAGCCCAGACATTCCGATTATACCCTTGGTGGCTCTTTTGCCTGAGACAAAATTTGGTGGCAGG	IIHIIHIIIIIIGEIIIIGIIFIIIIIGIGIIFGIEHIIHEIEIDGFIFCCIGGE@FBEEDGCBGEEEDGDEGDD	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10002:20694#CGATGT/1	99	chr7	63420623	0	25M3548N51M	=	63420635	12	ACTTTCGGCAGGATTAATGAGCCAGGGCAGTCTGCAGTGTTTTGTGGCCGTTCTGGAAAGCAGCTGAAACGATGTC	HHHHHHHGHHHHDHHGGGEEHHHGEBHHFHGHHHGHHEHEHHHHCCFCEF@B7B@;.=::===04@3=@7;;5=7:	NM:i:0	IH:i:1	HI:i:1	XS:A:+
HWUSI-EAS1737:4:100:10002:20694#CGATGT/2	147	chr7	63420635	82	13M3548N63M	=	63420623	-12	ATTAATGAGCCAGGGCAGTCTGCAGTGTTTTGTGGCCGTTCTGGAAAGCAGCTGAAACGATGTCACAGCAGTCAGC	BEEFBEC=BDBDGGEIBDBA>FFDFFAIHIFIDGIIIIHIIGIHIIIIIIFIIIIIHIIIIIIHIIIIIIIIIIII	NM:i:0	IH:i:1	HI:i:1	XS:A:+
HWUSI-EAS1737:4:100:10002:3249#ACAGTG/1	4	*	0	0	*	*	0	0	CTGTGGACTTATCACTGTCGCTGTCTGAGCTTGAACTGGGAGATCGGAAGAGCACACGTCTGAACTCCAGTCACAC	GIIIIIIIHIIIIGIIIIIDIIHIIHIIIIIIIIDIIIIIIFIHIIIIGIHIIIGIFIIIDHBIIGGIHHBHHHFH	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:3249#ACAGTG/2	4	*	0	0	*	*	0	0	CCCAGTTCAAGCTCAGACAGCGACAGTGATAAGTCCACAGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGA	IIIGHIIIGIIIIIIIIIIHIIIIIIIIGHIIIGIIIIIIHHHHIIGIIHIIHIHGGEIDEIII>BBDBDBFBHEE	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:3514#CGATGT/1	89	chr5	117565527	0	75M	*	0	0	GTGGGAGGAGAGAAGCTGGGAGCGAGAAAGCCAGGGCGAGCATTGCTCCGACCCGAGTGCGGAAATGAGAGACGC	?.=AAEFBBDBBDBD@@GEGHIGHHIHEEHIEBHIGIIIIGFIGDFIIFGDIIHIIIIIGGGGG@IIIFIIIGII	NM:i:0	IH:i:1	HI:i:1
HWUSI-EAS1737:4:100:10002:3514#CGATGT/2	4	*	0	0	*	*	0	0	TGGGGAAGCTCCCCAAGCGGAGCTAGGGGCATTAAAACAGTGGGAGGAGAGAAGCTGGGAGCGAGAAAGCCAGGGC	IIHIIIHIHIIIIIFGIIHHIIHFHIIHEGIIIIIDIBHIDGIFBGD@GDEEEDD3BBBBDHECFBE;AEGEDDDG	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:5277#ACAGTG/1	4	*	0	0	*	*	0	0	CGTCCTACTTGCTCTGGAGCCTGGGGCAGATGACTGCTGGAGCCCTGGAGTTCAAAGCTAGCCTGCGAGATCGGAA	IIIIEIIIHIIIIIIIIIIIIIIIIIIHIIGIIHIIIIHFHCIIHHIHEHDDEGGFGGGGGGEEGGGBGEBEEEBB	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:5277#ACAGTG/2	4	*	0	0	*	*	0	0	CGCAGGCTAGCTTTGAACTCCAGGGCTCCAGCAGTCATCTGCCCCAGGCTCCAGAGCAAGTAGGACGAGATCGGAA	IIIIIIIIIIIDIIIIIIIGIGHIHIHGIIIIIIIIHGHIIIIIIIIGGHIIIIFHHDCIDIGICGGEGGEGGHDG	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:9232#ACAGTG/1	4	*	0	0	*	*	0	0	CGCAGATACTCCTGTAAAAACTTACTGCTAATTCGTAGTGTAATAGGACCTTTCCTAGTGTTTTCAGCTTGTAACT	IIIIIIIIIIIIIIIIIIIIIIGIIIHIIIGIHIGIHIIIGHIIIHHFIIIIHIIHIIFHGHHHEGGGGIGEEGEG	IH:i:0	HI:i:0
HWUSI-EAS1737:4:100:10002:9232#ACAGTG/2	137	chr14	52698101	92	75M	*	0	0	CTCTACTATATTTAACCAATTTGTTCCCCTTTCAGTGTCATTGATTTCTGTGGTTCAAGGAAGAGTTTGCTAGCT	IIIIIIIGIIHIIIIIIIIIIIHIIIIIIIIIIIHIIIIIIIIIIIIIIIHIHHIIIIDHGIIFIGIIHIHHIIG	NM:i:0	IH:i:1	HI:i:1

I am unsure how to approach this new challenge. Any guidance will be much appreciated.

NOTE: The sample of lines from my sam file in my first post was derived using 'head'; while it appeared as though there was no whitespace between the sequence and the quality scores on same lines, that is actually an artifact of the 'head' process. The delimiters do actually exist.

NOTE: the sam file generated by MapSplice used both [space] and [tab] as field delimiters. My script included the sed command to replace [space] with [tab] so that DEXSeq would accept it.

NOTE: The original data come from fastq files generated from Illumina 1.5 paired-end reads with quality scores converted to sanger via Galaxy's FASTQ Groomer.

NOTE: While the script uses awk for the '.'->'N' replacement, standard awk accepts both [space] and [tab] as valid field delimiters and did not allow me to convert spaces to tabs, therefore I used sed for that step.

Pending a response, I will try to use Galaxy/Tophat bam output files converted to sam to see if they can be properly processed by the count script. However, I would prefer to use MapSplice output as it seems to be better geared for identifying differentially spliced junctions.

NOTE: Please pardon any misuse of genetics terminology. I am a mathematician/statistician/programmer, not a geneticist.

Best Regards,

Paul Bergmann

**areyes** · 09-18-2011, 04:21 AM

I see...

The flag term in the sam file is not correct if there are paired-end reads what you are analyzing. (2nd column) According to the sam format:

"0x1 template having multiple segments in sequencing"

in your case, there are some fields with 4 which would indicate that you have single reads. Because you are setting the parameter in dexseq_count.py saying that you have paired end reads, the script is complaining... MapSplice sam format seems a bit confusing. To be honest I have never use it.

Though I have use tophat and its works good with the script.

Pending a response, I will try to use Galaxy/Tophat bam output files converted to sam to see if they can be properly processed by the count script. However, I would prefer to use MapSplice output as it seems to be better geared for identifying differentially spliced junctions.

Have you try GSNAP? It also handles nicely this kind of alignments and I have not have any problem with its SAM output:

http://bioinformatics.oxfordjournals.org/content/27/18/2518.full

**FuzzyCoder** · 09-22-2011, 10:26 PM

Alejandro-

I have temporarily abandoned MapSplice and, after a brief failed attempt at using TopHat generated BAMs with DEXSeq, have spent the last week trying to arrive at the right set of parameters for running GSNAP. With my hardware, I am forced to use memory mapping rather than RAM allocation for the hash and genome.

My concern is that it will take an inordinate amount of time to align and map the the 6 sets of paired end reads. Each of the 12 FASTQ files is 2.9 - 3.6 GB. My computer is a Quad Core with 6 GB RAM. I used the option "-B 2" as anything higher kills the process or freezes the machine due to insufficient memory.

Can you provide me some idea of how long it will take to process two FASTQ files representing the paired end reads from a single replicate with GSNAP (each file ~3 GB). It seems to be quite a bit slower than MapSplice which took ~ 30 hours to process all 12 FASTQs (3 replicates per condition) at once.

Thank you.

I know you are the DEXSeq guru, and have only used GSNAP. However, my searches on the web have not reswulted in any meaningful information regarding actual runtime benchmarks for GSNAP, and I cannot wait weeks for the results.

**greenshell** · 09-27-2011, 08:52 AM

Please contact [email protected] for bug reports

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